Serine proteases

ABSTRACT

The present disclosure relates to serine proteases and variants thereof. Compositions containing the serine proteases are suitable for use in cleaning fabrics and hard surfaces, as well as in a variety of industrial applications.

The present disclosure relates to serine proteases cloned from Bacillusspp., and variants thereof. Compositions containing the serine proteasesare suitable for use in cleaning fabrics and hard surfaces, as well asin a variety of industrial applications.

Serine proteases are enzymes (EC No. 3.4.21) possessing an active siteserine that initiates hydrolysis of peptide bonds of proteins. There aretwo broad categories of serine proteases, based on their structure:chymotrypsin-like (trypsin-like) and subtilisin-like. The prototypicalsubtilisin (EC No. 3.4.21.62) was initially obtained from Bacillussubtilis. Subtilisins and their homologues are members of the S8peptidase family of the MEROPS classification scheme. Members of familyS8 have a catalytic triad in the order Asp, His and Ser in their aminoacid sequence.

Although serine proteases have long been known in the art of industrialenzymes, there remains a need for further serine proteases that aresuitable for particular conditions and uses.

The present compositions and methods relate to recombinant serineproteases cloned from Bacillus spp., and variants thereof. Compositionscontaining the serine proteases are suitable for use in cleaning fabricsand hard surfaces, as well as in a variety of industrial applications.

In some embodiments, the invention is a BspAP02013-clade of subtilisins.In some embodiments, the invention is a recombinant polypeptide oractive fragment thereof of a BspAP02013-clade subtilisin. In someembodiments, the BspAP02013-clade of subtilisins comprises a subtilisinor recombinant polypeptide or active fragment thereof comprising a DTGIXXXHXDLXXXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:11) motif, wherein theinitial D is the active site Aspartic acid, the terminal H is the activesite Histidine, and X is any amino acid (“Motif 1”); and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9. In other embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising aDTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:12) motif, whereinthe initial D is the active site Aspartic acid, the terminal H is theactive site Histidine, X is any amino acid, and X_(a) is T, S or F orX_(a) is S or F (“Motif 2”); and an amino acid sequence having at least80% amino acid sequence identity to an amino acid sequence of SEQ IDNO:3, 6, or 9. In still further embodiments, the BspAP02013-clade ofsubtilisins comprises a subtilisin or recombinant polypeptide or activefragment thereof comprising a DTGIXXXHXDLXXXXXGGXSVFT DSXXX_(b)XXXXDXXGH(SEQ ID NO:13) motif, wherein the initial D is the active site Asparticacid, the terminal H is the active site Histidine, X is any amino acid,and X_(b) is S or R or X_(b) is S (“Motif 3”); and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9. In yet another embodiment, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising a DTGIXXXHXDLXaXXXXGGXSVFTDSXXX_(b)XXXXDXXGH (SEQ ID NO:14) motif, wherein theinitial D is the active site Aspartic acid; the terminal H is the activesite Histidine; X is any amino acid; X_(a) is T, S or F or X_(a) is S orF; and X_(b) is S or R or X_(b) is S (“Motif 4”); and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9. In some embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising a DTGIXXXHXDLXANVXGGXSVFTDSANXDPFXDX XGH (SEQ ID NO:15) motif, wherein the initial D is theactive site Aspartic acid and the terminal H is the active siteHistidine, and X is any amino acid (“Motif 5”); and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9. In some embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising a DTGIXXXHXDLX_(a)ANVXGGXSVFTDSANXDPFXDXXGH (SEQ ID NO:20) motif, wherein theinitial D is the active site Aspartic acid; the terminal H is the activesite Histidine; X is any amino acid; and X_(a) is S or F (“Motif 6”);and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9. In someembodiments, the BspAP02013-clade of subtilisins comprises a subtilisinor recombinant polypeptide or active fragment thereof comprising aDTGIXXXHXDLXANVXGGXSVFTDSA NX_(b)DPFXDXXGH (SEQ ID NO:21) motif, whereinthe initial D is the active site Aspartic acid and the terminal H is theactive site Histidine, X is any amino acid, and X_(b) is S (“Motif 7”);and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9. In someembodiments, the BspAP02013-clade of subtilisins comprises a subtilisinor recombinant polypeptide or active fragment thereof comprising a DTGIXXXHXDLXXXXXGGXSVFXDXXXXXXXXDXXGH (SEQ ID NO:22) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid (“Motif 8”); and an aminoacid sequence having at least 80% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9. In some embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising aDTGIXXXHXDLXXNVXGGXSVFXDXXNXDPXXDXXGH (SEQ ID NO:23) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid (“Motif 9”); and an aminoacid sequence having at least 80% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9.

In some embodiments, the recombinant polypeptide or active fragmentthereof comprises an amino acid sequence having at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99% amino acid sequence identity to an amino acid sequence of SEQ IDNO:3, 6 or 9. In some embodiments, the recombinant polypeptide hasprotease activity, or subtilisin activity, specifically caseinhydrolysis activity. In some embodiments, the recombinant polypeptideretains at least 50% of its maximal protease activity at a pH range of 8to 12. In some embodiments, the recombinant polypeptide retains at least50% of its maximal protease activity at a temperature range of 55° C. to75° C. In some embodiments, the recombinant polypeptide has cleaningactivity in a detergent composition, including, for example, anautomatic dish washing detergent and a laundry detergent.

In some embodiments, the invention is a composition comprising asurfactant and the recombinant polypeptide or active fragment thereofdescribed herein. In some embodiments, the surfactant is selected fromthe group consisting of a non-ionic surfactant, an anionic surfactant, acationic surfactant, a zwitterionic surfactant, an ampholyticsurfactant, a semi-polar non-ionic surfactant, and a combinationthereof. In some embodiments, the composition is a detergentcomposition, such as a laundry detergent, a fabric softening detergent,a dishwashing detergent, and a hard-surface cleaning detergent. In someembodiments, the composition further comprises at least one calcium ionand/or zinc ion, at least one stabilizer, at least one bleaching agent,phosphate, or borate. In some embodiments the composition isphosphate-free and/or borate-free. In some embodiments, the compositionis a granular, powder, solid, bar, liquid, tablet, gel, paste or unitdose composition. In some embodiments, the composition furthercomprising one or more additional enzymes or enzyme derivatives selectedfrom the group consisting of acyl transferases, alpha-amylases,beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases,beta-galactosidases, carrageenases, catalases, cellobiohydrolases,cellulases, chondroitinases, cutinases, endo-beta-1, 4-glucanases,endo-beta-mannanases, esterases, exo-mannanases, galactanases,glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases,lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases,pectate lyases, pectin acetyl esterases, pectinases, pentosanases,peroxidases, phenoloxidases, phosphatases, phospholipases, phytases,polygalacturonases, proteases, pullulanases, reductases,rhamnogalacturonases, beta-glucanases, tannases, transglutaminases,xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases,metalloproteases, additional serine proteases, and combinations thereof.

Some embodiments are directed to a method of cleaning, comprisingcontacting a surface or an item with a recombinant polypeptide or activefragment thereof or a composition described herein. Other embodimentsare directed to a method for producing the recombinant polypeptide oractive fragment thereof comprising stably transforming a host cell withan expression vector comprising a polynucleotide encoding therecombinant polypeptide or active fragment thereof described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a plasmid map for expression of BspAP02013 protease.

FIG. 2 provides a plasmid map for expression of BspM02866 protease.

FIG. 3 provides a plasmid map for expression of BspZ00056 protease.

FIG. 4 provides a plot of protease activity of BspAP02013 protease onDMC substrate.

FIG. 5 provides a plot of protease activity of BspM02866 protease on DMCsubstrate.

FIG. 6 provides a plot of protease activity of BspZ00056 protease on DMCsubstrate.

FIGS. 7A to 7E provide CLUSTAL W (1.83) multiple sequence alignment ofsubtilisins including BspAP02013, BspM02866, and BspZ00056.

FIG. 8 provides phylogenetic tree of subtilisins including BspAP02013,BspM02866, and BspZ00056.

FIGS. 9A to 9C illustrate structural alignment of BspAP02013-cladesubtilisins (1-4) with subtilisin BPN′ from B. amyloliquefaciens (5) andthe subtilisin from B. lentus (6), where the common motif is shown by abox.

FIG. 10 illustrates the potential structural consequences of sequencemotif changes found in the BspAP02013-clade subtilisins.

Described are compositions and methods relating to recombinant serineproteases from Bacillus species. The compositions and methods are based,in part, on the observation that recombinant BspAP02013, BspM02866, andBspZ00056, among others, have protease activity in the presence of asurfactant, in basic reaction conditions, and at elevated temperatures.These features of BspAP02013, BspM02866, and BspZ00056 make theseproteases well suited for use in cleaning fabrics and hard surfaces, aswell as in textile, leather and feather processing. The new proteasesare also well suited to inclusion in compositions for proteindegradation, including but not limited to laundry and dishwashingdetergents.

Prior to describing the present compositions and methods in detail, thefollowing terms are defined for clarity. Terms and abbreviations notdefined should be accorded their ordinary meaning as used in the art.Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art. Unless otherwise indicated, the practice of thepresent disclosure involves conventional techniques commonly used inmolecular biology, protein engineering, and microbiology. Although anymethods and materials similar or equivalent to those described hereinfind use in the practice of the present disclosure, some suitablemethods and materials are described herein. The terms definedimmediately below are more fully described by reference to theSpecification as a whole.

As used herein, the singular “a,” “an” and “the” includes the pluralunless the context clearly indicates otherwise. Unless otherwiseindicated, nucleic acid sequences are written left to right in 5′ to 3′orientation; and amino acid sequences are written left to right in aminoto carboxy orientation. It is to be understood that this disclosure isnot limited to the particular methodology, protocols, and reagentsdescribed herein, absent an indication to the contrary.

It is intended that every maximum numerical limitation given throughoutthis Specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this Specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this Specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

As used herein in connection with a numerical value, the term “about”refers to a range of +/−0.5 of the numerical value, unless the term isotherwise specifically defined in context. For instance, the phrase a“pH value of about 6” refers to pH values of from 5.5 to 6.5, unless thepH value is specifically defined otherwise.

As used herein, the terms “protease” and “proteinase” refer to an enzymethat has the ability to break down proteins and peptides. A protease hasthe ability to conduct “proteolysis,” by hydrolysis of peptide bondsthat link amino acids together in a peptide or polypeptide chain formingthe protein. This activity of a protease as a protein-digesting enzymeis referred to as “proteolytic activity.” Many well-known proceduresexist for measuring proteolytic activity. For example, proteolyticactivity may be ascertained by comparative assays that analyze therespective protease's ability to hydrolyze a suitable substrate.Exemplary substrates useful in the analysis of protease or proteolyticactivity, include, but are not limited to, di-methyl casein (SigmaC-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625),and bovine keratin (ICN Biomedical 902111). Colorimetric assaysutilizing these substrates are well known in the art (See e.g.,WO99/34011 and U.S. Pat. No. 6,376,450). The pNA peptidyl assay (Seee.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) also finds use indetermining the active enzyme concentration. This assay measures therate at which p-nitroaniline is released as the enzyme hydrolyzes asoluble synthetic substrate, such assuccinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide(suc-AAPF-pNA). The rate of production of yellow color from thehydrolysis reaction is measured at 410 nm on a spectrophotometer and isproportional to the active enzyme concentration. In addition, absorbancemeasurements at 280 nanometers (nm) can be used to determine the totalprotein concentration in a sample of purified protein. The activity onsubstrate/protein concentration gives the enzyme specific activity.

The term “variant,” with respect to a polypeptide, refers to apolypeptide that differs from a specified wild-type, parental, orreference polypeptide in that it includes one or morenaturally-occurring or man-made substitutions, insertions, or deletionsof an amino acid. Similarly, the term “variant,” with respect to apolynucleotide, refers to a polynucleotide that differs in nucleotidesequence from a specified wild-type, parental, or referencepolynucleotide. The identity of the wild-type, parental, or referencepolypeptide or polynucleotide will be apparent from context.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii,and B. thuringiensis. It is recognized that the genus Bacillus continuesto undergo taxonomical reorganization. Thus, it is intended that thegenus include species that have been reclassified, including but notlimited to such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus”, or B. polymyxa, which is now“Paenibacillus polymyxa” The production of resistant endospores understressful environmental conditions is considered the defining feature ofthe genus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

As used herein, the term “mutation” refers to changes made to areference amino acid or nucleic acid sequence. It is intended that theterm encompass substitutions, insertions and deletions.

As used herein, the term “vector” refers to a nucleic acid constructused to introduce or transfer nucleic acid(s) into a target cell ortissue. A vector is typically used to introduce foreign DNA into a cellor tissue. Vectors include plasmids, cloning vectors, bacteriophages,viruses (e.g., viral vector), cosmids, expression vectors, shuttlevectors, and the like. A vector typically includes an origin ofreplication, a multicloning site, and a selectable marker. The processof inserting a vector into a target cell is typically referred to astransformation. The present invention includes, in some embodiments, avector that comprises a DNA sequence encoding a serine proteasepolypeptide (e.g., precursor or mature serine protease polypeptide) thatis operably linked to a suitable prosequence (e.g., secretory, signalpeptide sequence, etc.) capable of effecting the expression of the DNAsequence in a suitable host, and the folding and translocation of therecombinant polypeptide chain.

As used herein in the context of introducing a nucleic acid sequenceinto a cell, the term “introduced” refers to any method suitable fortransferring the nucleic acid sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, electroporation, conjugation, andtransduction. Transformation refers to the genetic alteration of a cellwhich results from the uptake, optional genomic incorporation, andexpression of genetic material (e.g., DNA).

As used herein, a nucleic acid is “operably linked” with another nucleicacid sequence when it is placed into a functional relationship withanother nucleic acid sequence. For example, a promoter or enhancer isoperably linked to a nucleotide coding sequence if the promoter affectsthe transcription of the coding sequence. A ribosome binding site may beoperably linked to a coding sequence if it is positioned so as tofacilitate translation of the coding sequence. Typically, “operablylinked” DNA sequences are contiguous. However, enhancers do not have tobe contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers may be used in accordance with conventionalpractice.

As used herein the term “gene” refers to a polynucleotide (e.g., a DNAsegment), that encodes a polypeptide and includes regions preceding andfollowing the coding regions. In some instances a gene includesintervening sequences (introns) between individual coding segments(exons).

As used herein, “recombinant” when used with reference to a celltypically indicates that the cell has been modified by the introductionof a foreign nucleic acid sequence or that the cell is derived from acell so modified. For example, a recombinant cell may comprise a genenot found in identical form within the native (non-recombinant) form ofthe cell, or a recombinant cell may comprise a native gene (found in thenative form of the cell) that has been modified and re-introduced intothe cell. A recombinant cell may comprise a nucleic acid endogenous tothe cell that has been modified without removing the nucleic acid fromthe cell; such modifications include those obtained by gene replacement,site-specific mutation, and related techniques known to those ofordinary skill in the art. Recombinant DNA technology includestechniques for the production of recombinant DNA in vitro and transferof the recombinant DNA into cells where it may be expressed orpropagated, thereby producing a recombinant polypeptide. “Recombination”and “recombining” of polynucleotides or nucleic acids refer generally tothe assembly or combining of two or more nucleic acid or polynucleotidestrands or fragments to generate a new polynucleotide or nucleic acid.

A nucleic acid or polynucleotide is said to “encode” a polypeptide if,in its native state or when manipulated by methods known to those ofskill in the art, it can be transcribed and/or translated to produce thepolypeptide or a fragment thereof. The anti-sense strand of such anucleic acid is also said to encode the sequence.

The terms “host strain” and “host cell” refer to a suitable host for anexpression vector comprising a DNA sequence of interest.

A “protein” or “polypeptide” comprises a polymeric sequence of aminoacid residues. The terms “protein” and “polypeptide” are usedinterchangeably herein. The single and 3-letter code for amino acids asdefined in conformity with the IUPAC-IUB Joint Commission on BiochemicalNomenclature (JCBN) is used throughout this disclosure. The singleletter X refers to any of the twenty amino acids. It is also understoodthat a polypeptide may be coded for by more than one nucleotide sequencedue to the degeneracy of the genetic code. Mutations can be named by theone letter code for the parent amino acid, followed by a position numberand then the one letter code for the variant amino acid. For example,mutating glycine (G) at position 87 to serine (S) is represented as“G087S” or “G87S. When describing modifications, a position followed byamino acids listed in parentheses indicates a list of substitutions atthat position by any of the listed amino acids. For example, 6(L,I)means position 6 can be substituted with a leucine or isoleucine. Attimes, in a sequence, a slash (/) is used to define substitutions, e.g.F/V, indicates that the particular position may have a phenylalanine orvaline at that position.

A “prosequence” or “propeptide sequence” refers to an amino acidsequence between the signal peptide sequence and mature proteasesequence that is necessary for the proper folding and secretion of theprotease; they are sometimes referred to as intramolecular chaperones.Cleavage of the prosequence or propeptide sequence results in a matureactive protease. Bacterial serine proteases are often expressed aspro-enzymes.

The terms “signal sequence” and “signal peptide” refer to a sequence ofamino acid residues that may participate in the secretion or directtransport of the mature or precursor form of a protein. The signalsequence is typically located N-terminal to the precursor or matureprotein sequence. The signal sequence may be endogenous or exogenous. Asignal sequence is normally absent from the mature protein. A signalsequence is typically cleaved from the protein by a signal peptidaseafter the protein is transported.

The term “mature” form of a protein, polypeptide, or peptide refers tothe functional form of the protein, polypeptide, or peptide without thesignal peptide sequence and propeptide sequence.

The term “precursor” form of a protein or peptide refers to a matureform of the protein having a prosequence operably linked to the amino orcarbonyl terminus of the protein. The precursor may also have a “signal”sequence operably linked to the amino terminus of the prosequence. Theprecursor may also have additional polypeptides that are involved inpost-translational activity (e.g., polypeptides cleaved therefrom toleave the mature form of a protein or peptide).

The term “wild-type” in reference to an amino acid sequence or nucleicacid sequence indicates that the amino acid sequence or nucleic acidsequence is a native or naturally-occurring sequence. As used herein,the term “naturally-occurring” refers to anything (e.g., proteins, aminoacids, or nucleic acid sequences) that is found in nature. Conversely,the term “non-naturally occurring” refers to anything that is not foundin nature (e.g., recombinant nucleic acids and protein sequencesproduced in the laboratory or modification of the wild-type sequence).

As used herein with regard to amino acid residue positions,“corresponding to” or “corresponds to” or “corresponds” refers to anamino acid residue at the enumerated position in a protein or peptide,or an amino acid residue that is analogous, homologous, or equivalent toan enumerated residue in a protein or peptide. As used herein,“corresponding region” generally refers to an analogous position in arelated proteins or a reference protein.

The terms “derived from” and “obtained from” refer to not only a proteinproduced or producible by a strain of the organism in question, but alsoa protein encoded by a DNA sequence isolated from such strain andproduced in a host organism containing such DNA sequence. Additionally,the term refers to a protein which is encoded by a DNA sequence ofsynthetic and/or cDNA origin and which has the identifyingcharacteristics of the protein in question. To exemplify, “proteasesderived from Bacillus” refers to those enzymes having proteolyticactivity that are naturally produced by Bacillus, as well as to serineproteases like those produced by Bacillus sources but which through theuse of genetic engineering techniques are produced by other host cellstransformed with a nucleic acid encoding the serine proteases.

The term “identical” in the context of two polynucleotide or polypeptidesequences refers to the nucleic acids or amino acids in the twosequences that are the same when aligned for maximum correspondence, asmeasured using sequence comparison or analysis algorithms describedbelow and known in the art.

As used herein, “% identity” or percent identity” or “PID” refers toprotein sequence identity. Percent identity may be determined usingstandard techniques known in the art. Useful algorithms include theBLAST algorithms (See, Altschul et al., J Mol Biol, 215:403-410, 1990;and Karlin and Altschul, Proc Natl Acad Sci USA, 90:5873-5787, 1993).The BLAST program uses several search parameters, most of which are setto the default values. The NCBI BLAST algorithm finds the most relevantsequences in terms of biological similarity but is not recommended forquery sequences of less than 20 residues (Altschul et al., Nucleic AcidsRes, 25:3389-3402, 1997; and Schaffer et al., Nucleic Acids Res,29:2994-3005, 2001). Exemplary default BLAST parameters for a nucleicacid sequence searches include: Neighboring words threshold=11; E-valuecutoff=10; Scoring Matrix=NUC.3.1 (match=1, mismatch=−3); Gap Opening=5;and Gap Extension=2. Exemplary default BLAST parameters for amino acidsequence searches include: Word size=3; E-value cutoff=10; ScoringMatrix=BLOSUM62; Gap Opening=11; and Gap extension=1. A percent (%)amino acid sequence identity value is determined by the number ofmatching identical residues divided by the total number of residues ofthe “reference” sequence including any gaps created by the program foroptimal/maximum alignment. BLAST algorithms refer to the “reference”sequence as the “query” sequence.

As used herein, “homologous proteins” or “homologous proteases” refersto proteins that have distinct similarity in primary, secondary, and/ortertiary structure. Protein homology can refer to the similarity inlinear amino acid sequence when proteins are aligned. Homologous searchof protein sequences can be done using BLASTP and PSI-BLAST from NCBIBLAST with threshold (E-value cut-off) at 0.001. (Altschul S F, Madde TL, Shaffer A A, Zhang J, Zhang Z, Miller W, Lipman D J. Gapped BLAST andPSI BLAST a new generation of protein database search programs. NucleicAcids Res 1997 Set 1; 25(17):3389-402). Using this information, proteinssequences can be grouped. A phylogenetic tree can be built using theamino acid sequences. Amino acid sequences can be entered in a programsuch as the Vector NTI Advance suite and a Guide Tree can be createdusing the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol,4:406-425, 1987). The tree construction can be calculated using Kimura'scorrection for sequence distance and ignoring positions with gaps. Aprogram such as AlignX can display the calculated distance values inparenthesis following the molecule name displayed on the phylogenetictree.

Understanding the homology between molecules can reveal the evolutionaryhistory of the molecules as well as information about their function; ifa newly sequenced protein is homologous to an already characterizedprotein, there is a strong indication of the new protein's biochemicalfunction. The most fundamental relationship between two entities ishomology; two molecules are said to be homologous if they have beenderived from a common ancestor. Homologous molecules, or homologs, canbe divided into two classes, paralogs and orthologs. Paralogs arehomologs that are present within one species. Paralogs often differ intheir detailed biochemical functions. Orthologs are homologs that arepresent within different species and have very similar or identicalfunctions. A protein superfamily is the largest grouping (clade) ofproteins for which common ancestry can be inferred. Usually this commonancestry is based on sequence alignment and mechanistic similarity.Superfamilies typically contain several protein families which showsequence similarity within the family. The term “protein clan” iscommonly used for protease superfamilies based on the MEROPS proteaseclassification system.

The CLUSTAL W algorithm is another example of a sequence alignmentalgorithm (See, Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994).Default parameters for the CLUSTAL W algorithm include: Gap openingpenalty=10.0; Gap extension penalty=0.05; Protein weight matrix=BLOSUMseries; DNA weight matrix=IUB; Delay divergent sequences %=40; Gapseparation distance=8; DNA transitions weight=0.50; List hydrophilicresidues=GPSNDQEKR; Use negative matrix=OFF; Toggle Residue specificpenalties=ON; Toggle hydrophilic penalties=ON; and Toggle end gapseparation penalty=OFF. In CLUSTAL algorithms, deletions occurring ateither terminus are included. For example, a variant with a five aminoacid deletion at either terminus (or within the polypeptide) of apolypeptide of 500 amino acids would have a percent sequence identity of99% (495/500 identical residues x 100) relative to the “reference”polypeptide. Such a variant would be encompassed by a variant having “atleast 99% sequence identity” to the polypeptide.

A nucleic acid or polynucleotide is “isolated” when it is at leastpartially or completely separated from other components, including butnot limited to for example, other proteins, nucleic acids, cells, etc.Similarly, a polypeptide, protein or peptide is “isolated” when it is atleast partially or completely separated from other components, includingbut not limited to for example, other proteins, nucleic acids, cells,etc. On a molar basis, an isolated species is more abundant than areother species in a composition. For example, an isolated species maycomprise at least about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100% (on amolar basis) of all macromolecular species present. Preferably, thespecies of interest is purified to essential homogeneity (i.e.,contaminant species cannot be detected in the composition byconventional detection methods). Purity and homogeneity can bedetermined using a number of techniques well known in the art, such asagarose or polyacrylamide gel electrophoresis of a nucleic acid or aprotein sample, respectively, followed by visualization upon staining.If desired, a high-resolution technique, such as high performance liquidchromatography (HPLC) or a similar means can be utilized forpurification of the material.

The term “purified” as applied to nucleic acids or polypeptidesgenerally denotes a nucleic acid or polypeptide that is essentially freefrom other components as determined by analytical techniques well knownin the art (e.g., a purified polypeptide or polynucleotide forms adiscrete band in an electrophoretic gel, chromatographic eluate, and/ora media subjected to density gradient centrifugation). For example, anucleic acid or polypeptide that gives rise to essentially one band inan electrophoretic gel is “purified.” A purified nucleic acid orpolypeptide is at least about 50% pure, usually at least about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%or more pure (e.g., percent by weight on a molar basis). In a relatedsense, a composition is enriched for a molecule when there is asubstantial increase in the concentration of the molecule afterapplication of a purification or enrichment technique. The term“enriched” refers to a compound, polypeptide, cell, nucleic acid, aminoacid, or other specified material or component that is present in acomposition at a relative or absolute concentration that is higher thana starting composition.

As used herein, the term “functional assay” refers to an assay thatprovides an indication of a protein's activity. In some embodiments, theterm refers to assay systems in which a protein is analyzed for itsability to function in its usual capacity. For example, in the case of aprotease, a functional assay involves determining the effectiveness ofthe protease to hydrolyze a proteinaceous substrate.

The term “cleaning activity” refers to a cleaning performance achievedby a serine protease polypeptide or reference protease under conditionsprevailing during the proteolytic, hydrolyzing, cleaning, or otherprocess of the disclosure. In some embodiments, cleaning performance ofa serine protease polypeptide or reference protease may be determined byusing various assays for cleaning one or more various enzyme sensitivestains on an item or surface (e.g., a stain resulting from food, grass,blood, ink, milk, oil, and/or egg protein). Cleaning performance of avariant or reference protease can be determined by subjecting the stainon the item or surface to standard wash condition(s) and assessing thedegree to which the stain is removed by using various chromatographic,spectrophotometric, or other quantitative methodologies. Exemplarycleaning assays and methods are known in the art and include, but arenot limited to those described in WO99/34011 and U.S. Pat. No.6,605,458, both of which are herein incorporated by reference, as wellas those cleaning assays and methods included in the Examples providedbelow.

The term “cleaning effective amount” of a serine protease polypeptide orreference protease refers to the amount of protease that achieves adesired level of enzymatic activity in a specific cleaning composition.Such effective amounts are readily ascertained by one of ordinary skillin the art and are based on many factors, such as the particularprotease used, the cleaning application, the specific composition of thecleaning composition, and whether a liquid or dry (e.g., granular,tablet, bar) composition is required, etc.

The term “cleaning adjunct material” refers to any liquid, solid, orgaseous material included in cleaning composition other than a serineprotease polypeptide of the disclosure. In some embodiments, thecleaning compositions of the present disclosure include one or morecleaning adjunct materials. Each cleaning adjunct material is typicallyselected depending on the particular type and form of cleaningcomposition (e.g., liquid, granule, powder, bar, paste, spray, tablet,gel, foam, or other composition). Preferably, each cleaning adjunctmaterial is compatible with the protease enzyme used in the composition.

Cleaning compositions and cleaning formulations include any compositionthat is suited for cleaning, bleaching, disinfecting, and/or sterilizingany object, item, and/or surface. Such compositions and formulationsinclude, but are not limited to for example, liquid and/or solidcompositions, including cleaning or detergent compositions (e.g.,liquid, tablet, gel, bar, granule, and/or solid laundry cleaning ordetergent compositions and fine fabric detergent compositions; hardsurface cleaning compositions and formulations, such as for glass, wood,ceramic and metal counter tops and windows; carpet cleaners; ovencleaners; fabric fresheners; fabric softeners; and textile, laundrybooster cleaning or detergent compositions, laundry additive cleaningcompositions, and laundry pre-spotter cleaning compositions; dishwashingcompositions, including hand or manual dishwashing compositions (e.g.,“hand” or “manual” dishwashing detergents) and automatic dishwashingcompositions (e.g., “automatic dishwashing detergents”). Single dosageunit forms also find use with the present invention, including but notlimited to pills, tablets, gelcaps, or other single dosage units such aspre-measured powders or liquids.

Cleaning composition or cleaning formulations, as used herein, include,unless otherwise indicated, granular or powder-form all-purpose orheavy-duty washing agents, especially cleaning detergents; liquid,granular, gel, solid, tablet, paste, or unit dosage form all-purposewashing agents, especially the so-called heavy-duty liquid (HDL)detergent or heavy-duty dry (HDD) detergent types; liquid fine-fabricdetergents; hand or manual dishwashing agents, including those of thehigh-foaming type; hand or manual dishwashing, automatic dishwashing, ordishware or tableware washing agents, including the various tablet,powder, solid, granular, liquid, gel, and rinse-aid types for householdand institutional use; liquid cleaning and disinfecting agents,including antibacterial hand-wash types, cleaning bars, mouthwashes,denture cleaners, car shampoos, carpet shampoos, bathroom cleaners; hairshampoos and/or hair-rinses for humans and other animals; shower gelsand foam baths and metal cleaners; as well as cleaning auxiliaries, suchas bleach additives and “stain-stick” or pre-treat types. In someembodiments, granular compositions are in “compact” form; in someembodiments, liquid compositions are in a “concentrated” form.

As used herein, “fabric cleaning compositions” include hand and machinelaundry detergent compositions including laundry additive compositionsand compositions suitable for use in the soaking and/or pretreatment ofstained fabrics (e.g., clothes, linens, and other textile materials).

As used herein, “non-fabric cleaning compositions” include non-textile(i.e., non-fabric) surface cleaning compositions, including, but notlimited to for example, hand or manual or automatic dishwashingdetergent compositions, oral cleaning compositions, denture cleaningcompositions, contact lens cleaning compositions, wound debridementcompositions, and personal cleansing compositions.

As used herein, the term “detergent composition” or “detergentformulation” is used in reference to a composition intended for use in awash medium for the cleaning of soiled or dirty objects, includingparticular fabric and/or non-fabric objects or items. Such compositionsof the present disclosure are not limited to any particular detergentcomposition or formulation. Indeed, in some embodiments, the detergentsof the disclosure comprise at least one serine protease polypeptide ofthe disclosure and, in addition, one or more surfactants,transferase(s), hydrolytic enzymes, oxido reductases, builders (e.g., abuilder salt), bleaching agents, bleach activators, bluing agents,fluorescent dyes, caking inhibitors, masking agents, enzyme activators,antioxidants, and/or solubilizers. In some instances, a builder salt isa mixture of a silicate salt and a phosphate salt, preferably with moresilicate (e.g., sodium metasilicate) than phosphate (e.g., sodiumtripolyphosphate). Some compositions of the disclosure, such as, but notlimited to, cleaning compositions or detergent compositions, do notcontain any phosphate (e.g., phosphate salt or phosphate builder).

As used herein, the term “bleaching” refers to the treatment of amaterial (e.g., fabric, laundry, pulp, etc.) or surface for a sufficientlength of time and/or under appropriate pH and/or temperature conditionsto effect a brightening (i.e., whitening) and/or cleaning of thematerial. Examples of chemicals suitable for bleaching include, but arenot limited to, for example, ClO₂, H₂O₂, peracids, NO₂, etc.

As used herein, “wash performance” of a protease (e.g., a serineprotease polypeptide of the disclosure) refers to the contribution of aserine protease polypeptide to washing that provides additional cleaningperformance to the detergent as compared to the detergent without theaddition of the serine protease polypeptide to the composition. Washperformance is compared under relevant washing conditions. In some testsystems, other relevant factors, such as detergent composition, sudconcentration, water hardness, washing mechanics, time, pH, and/ortemperature, can be controlled in such a way that condition(s) typicalfor household application in a certain market segment (e.g., hand ormanual dishwashing, automatic dishwashing, dishware cleaning, tablewarecleaning, fabric cleaning, etc.) are imitated.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,sud concentration, type of detergent and water hardness, actually usedin households in a hand dishwashing, automatic dishwashing, or laundrydetergent market segment.

As used herein, the term “disinfecting” refers to the removal ofcontaminants from the surfaces, as well as the inhibition or killing ofmicrobes on the surfaces of items. It is not intended that the presentdisclosure be limited to any particular surface, item, or contaminant(s)or microbes to be removed.

The “compact” form of the cleaning compositions herein is best reflectedby density and, in terms of composition, by the amount of inorganicfiller salt. Inorganic filler salts are conventional ingredients ofdetergent compositions in powder form. In conventional detergentcompositions, the filler salts are present in substantial amounts,typically about 17 to about 35% by weight of the total composition. Incontrast, in compact compositions, the filler salt is present in amountsnot exceeding about 15% of the total composition. In some embodiments,the filler salt is present in amounts that do not exceed about 10%, ormore preferably, about 5%, by weight of the composition. In someembodiments, the inorganic filler salts are selected from the alkali andalkaline-earth-metal salts of sulfates and chlorides. In someembodiments, the filler salt is sodium sulfate.

The present disclosure provides novel serine protease enzymes. Theserine protease polypeptides of the present disclosure include isolated,recombinant, substantially pure, or non-naturally occurringpolypeptides. In some embodiments, the polypeptides are useful incleaning applications and can be incorporated into cleaning compositionsthat are useful in methods of cleaning an item or a surface in needthereof.

The BspAP02013-clade of subtilisins are characterized by two separate 2amino acid residue insertions; Insertion 1 is after conserved positionsHPDL at positions 39-42 and Insertion 2 is after position N56, whereinthe amino acid positions are numbered by correspondence with the aminoacid sequence of subtilisin BPN′, wherein both insertions occur in thespan of residues linking the catalytic aspartic acid (D32) and catalytichistidine (H68) that form part of the characteristic catalytic triad.

In some embodiments, the invention is a BspAP02013-clade of subtilisins.In some embodiments, the invention is a recombinant polypeptide oractive fragment thereof of the BspAP02013-clade. In some embodiments,the BspAP02013-clade of subtilisins comprises a subtilisin orrecombinant polypeptide or active fragment thereof comprising aDTGIXXXHXDL XXXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:11) motif, wherein theinitial D is the active site Aspartic acid, the terminal H is the activesite Histidine, and X is any amino acid (“Motif 1”); and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9. In other embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising aDTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:12) motif, whereinthe initial D is the active site Aspartic acid, the terminal H is theactive site Histidine, X is any amino acid, and X_(a) is T, S or F orX_(a) is S or F (“Motif 2”); and an amino acid sequence having at least80% amino acid sequence identity to an amino acid sequence of SEQ IDNO:3, 6, or 9. In still further embodiments, the BspAP02013-clade ofsubtilisins comprises a subtilisin or recombinant polypeptide or activefragment thereof comprising a DTGIXXXHXDL XXXXXGGXSVFTDSXXX_(b)XXXXDXXGH(SEQ ID NO:13) motif, wherein the initial D is the active site Asparticacid, the terminal H is the active site Histidine, X is any amino acid,and X_(b) is S or R or X_(b) is S (“Motif 3”); and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9. In yet other embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising aDTGIXXXHXDLX_(a)XXXXGGXSVFTDSX XX_(b)XXXXDXXGH (SEQ ID NO:14), whereinthe initial D is the active site Aspartic acid; the terminal H is theactive site Histidine; X is any amino acid; X_(a) is T, S or F or X_(a)is S or F; and X_(b) is S or R or X_(b) is S (“Motif 4”); and an aminoacid sequence having at least 80% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9. In some embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising aDTGIXXXHXDLXANVXGGXSVFTDSANXDPFXDXXGH (SEQ ID NO:15) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid (“Motif 5”); and an aminoacid sequence having at least 80% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9. In some embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising aDTGIXXXHXDLX_(a)ANVXG GXSVFTDSANXDPFXDXXGH (SEQ ID NO:20) motif, whereinthe initial D is the active site Aspartic acid; the terminal H is theactive site Histidine; X is any amino acid; and X_(a) is S or F (“Motif6”); and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9. In someembodiments, the BspAP02013-clade of subtilisins comprises a subtilisinor recombinant polypeptide or active fragment thereof comprising aDTGIXXXHXDLXANVXGGXSVFTDSANX_(b)DPFXDXXGH (SEQ ID NO:21) motif, whereinthe initial D is the active site Aspartic acid and the terminal H is theactive site Histidine, X is any amino acid, and X_(b) is S (“Motif 7”);and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9. In someembodiments, the BspAP02013-clade of subtilisins comprises a subtilisinor recombinant polypeptide or active fragment thereof comprising aDTGIXXXHXDLXXXXXGGXSVFXDX XXXXXXXDXXGH (SEQ ID NO:22) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid (“Motif 8”); and an aminoacid sequence having at least 80% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9. In some embodiments, theBspAP02013-clade of subtilisins comprises a subtilisin or recombinantpolypeptide or active fragment thereof comprising a DTGI XXXHXDLXXNVXGGXSVFXDXXNXDPXXDXXGH (SEQ ID NO:23) motif, wherein the initial D is theactive site Aspartic acid and the terminal H is the active siteHistidine, and X is any amino acid (“Motif 9”); and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9.

In some embodiments, a subtilisin of the BspAP02013-clade of subtilisinsand/or the recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises an HX DLXANVXGGXS (SEQ ID NO:16) motif(“Sub-motif 1”), wherein the initial D is the active site Aspartic acid,the terminal GGXS is in the outermost strand of the central beta sheet,and X is any amino acid; and an amino acid sequence having at least 80%amino acid sequence identity to an amino acid sequence of SEQ ID NO:3,6, or 9. Sub-motif 1 includes Insertion 1. In some embodiments, asubtilisin of the BspAP02013-clade of subtilisins and/or the recombinantpolypeptide or active fragment thereof of the BspAP02013-clade comprisesan GGXSVFTDSA NXDPFXD (SEQ ID NO:17) motif (“Sub-motif 2”), wherein theinitial GGXS is in the outermost strand of the central beta sheet and Xis any amino acid; and an amino acid sequence having at least 80% aminoacid sequence identity to an amino acid sequence of SEQ ID NO:3, 6, or9. Sub-motif 2 includes Insertion 2. In some embodiments, a subtilisinof the BspAP02013-clade of subtilisins and/or the recombinantpolypeptide or active fragment thereof of the BspAP02013-clade comprisesan HXDLX_(a)ANVXGGXS (SEQ ID NO:18) motif (“Sub-motif 3”), wherein theinitial D is the active site Aspartic acid; the terminal GGXS is in theoutermost strand of the central beta sheet; X_(a) is T, S or F or X_(a)is S or F; and X is any amino acid. Sub-motif 3 includes Insertion 1;and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9. In someembodiments, a subtilisin of the BspAP02013-clade of subtilisins and/orthe recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises an GGXSVFTDSANX_(b)DPFXD (SEQ ID NO:19) motif(“Sub-motif 4”), wherein the initial GGXS is in the outermost strand ofthe central beta sheet, X is any amino acid, and X_(b) is S or R orX_(b) is S; and an amino acid sequence having at least 80% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9.Sub-motif 4 includes Insertion 2.

In some embodiments, a subtilisin of the BspAP02013-clade of subtilisinsand/or recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises a DTGI XXXHXDLXXXXXGGXSVFTDSXXXXXXXDXXGH (SEQID NO:11) motif, wherein the initial D is the active site Aspartic acid,the terminal H is the active site Histidine, and X is any amino acid;and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9, with theproviso that the subtilisin of the BspAP02013-clade of subtilisinsand/or recombinant polypeptide or active fragment thereof of theBspAP02013-clade does not comprise WP_012957833, ERN55058, orWP_022626565. In other embodiments, a subtilisin of the BspAP02013-cladeof subtilisins and/or recombinant polypeptide or active fragment thereofof the BspAP02013-clade comprises a DTGIXXXHXDLXXXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:11) motif, wherein the initial Dis the active site Aspartic acid, the terminal H is the active siteHistidine, and X is any amino acid; and an amino acid sequence having atleast 80% amino acid sequence identity to an amino acid sequence of SEQID NO:3, 6, or 9, with the proviso that the subtilisin of theBspAP02013-clade of subtilisins and/or recombinant polypeptide or activefragment thereof of the BspAP02013-clade does not comprise WP_012957833,ERN55058, WP_022626565, or WP_047973355.

In yet another embodiment, a subtilisin of the BspAP02013-clade ofsubtilisins and/or the recombinant polypeptide or active fragmentthereof of the BspAP02013-clade comprises aDTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:12) motif, whereinthe initial D is the active site Aspartic acid, the terminal H is theactive site Histidine, X is any amino acid, and X_(a) is T, S or F; andan amino acid sequence having at least 80% amino acid sequence identityto an amino acid sequence of SEQ ID NO:3, 6, or 9, with the proviso thatthe subtilisin of the BspAP02013-clade of subtilisins and/or recombinantpolypeptide or active fragment thereof of the BspAP02013-clade does notcomprise WP_012957833, ERN55058, or WP_022626565. In yet anotherembodiment, a subtilisin of the BspAP02013-clade of subtilisins and/orthe recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises a DTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXXXXXXDXXGH(SEQ ID NO:12) motif, wherein the initial D is the active site Asparticacid, the terminal H is the active site Histidine, X is any amino acid,and X_(a) is T, S or F; and an amino acid sequence having at least 80%amino acid sequence identity to an amino acid sequence of SEQ ID NO:3,6, or 9, with the proviso that the subtilisin of the BspAP02013-clade ofsubtilisins and/or recombinant polypeptide or active fragment thereof ofthe BspAP02013-clade does not comprise WP_012957833, ERN55058,WP_022626565, or WP_047973355. In yet a further embodiment, a subtilisinof the BspAP02013-clade of subtilisins and/or the recombinantpolypeptide or active fragment thereof of the BspAP02013-clade comprisesa DTGIXXXHXDLX_(a)XXXXGGXSVFT DSXXXXXXXD XXGH (SEQ ID NO:12) motif,wherein the initial D is the active site Aspartic acid, the terminal His the active site Histidine, X is any amino acid, and X_(a) is S or F;and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9, with theproviso that the subtilisin of the BspAP02013-clade of subtilisinsand/or recombinant polypeptide or active fragment thereof of theBspAP02013-clade does not comprise WP_047973355.

In still further embodiments, a subtilisin of the BspAP02013-clade ofsubtilisins and/or the recombinant polypeptide or active fragmentthereof of the BspAP02013-clade comprises aDTGIXXXHXDLXXXXXGGXSVFTDSXXX_(b)XXXXDXXGH (SEQ ID NO:13) motif, whereinthe initial D is the active site Aspartic acid, the terminal H is theactive site Histidine, X is any amino acid, and X_(b) is S or R; and anamino acid sequence having at least 80% amino acid sequence identity toan amino acid sequence of SEQ ID NO:3, 6, or 9, with the proviso thatthe subtilisin of the BspAP02013-clade of subtilisins and/or recombinantpolypeptide or active fragments thereof of the BspAP02013-clade does notcomprise WP_012957833, ERN55058, or WP_022626565. In yet anotherembodiment, a subtilisin of the BspAP02013-clade of subtilisins and/orthe recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises a DTGIXXXHXDLXXXXXGGXSVFTDSXXX_(b)XXXXD XXGH(SEQ ID NO:13) motif, wherein the initial D is the active site Asparticacid, the terminal H is the active site Histidine, X is any amino acid,and X_(b) is S or R; and an amino acid sequence having at least 80%amino acid sequence identity to an amino acid sequence of SEQ ID NO:3,6, or 9, with the proviso that the subtilisin of the BspAP02013-clade ofsubtilisins and/or recombinant polypeptide or active fragment thereof ofthe BspAP02013-clade does not comprise WP_012957833, ERN55058,WP_022626565, or WP_047973355. In yet still further embodiments, asubtilisin of the BspAP02013-clade of subtilisins and/or the recombinantpolypeptide or active fragment thereof of the BspAP02013-clade comprisesa DTGIXXXHXDL XXXXXGGXSVFTDSXXX_(b)XXXXDXXGH (SEQ ID NO:13) motif,wherein the initial D is the active site Aspartic acid, the terminal His the active site Histidine, X is any amino acid, and X_(b) is S; andan amino acid sequence having at least 80% amino acid sequence identityto an amino acid sequence of SEQ ID NO:3, 6, or 9, with the proviso thatthe subtilisin of the BspAP02013-clade of subtilisins and/or recombinantpolypeptide or active fragment thereof of the BspAP02013-clade does notcomprise WP_047973355.

In further embodiments, a subtilisin of the BspAP02013-clade ofsubtilisins and/or the recombinant polypeptide or active fragmentthereof of the BspAP02013-clade comprises aDTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXX_(b)XXXXDXXGH (SEQ ID NO:14), whereinthe initial D is the active site Aspartic acid; the terminal H is theactive site Histidine; X is any amino acid; X_(a) is T, S or F or X_(a)is S or F; and X_(b) is S or R or X_(b) is S; and an amino acid sequencehaving at least 80% amino acid sequence identity to an amino acidsequence of SEQ ID NO:3, 6, or 9, with the proviso that the subtilisinof the BspAP02013-clade of subtilisins and/or recombinant polypeptide oractive fragment thereof of the BspAP02013-clade does not compriseWP_012957833, ERN55058, or WP_022626565. In still further embodiments, asubtilisin of the BspAP02013-clade of subtilisins and/or the recombinantpolypeptide or active fragment thereof of the BspAP02013-clade comprisesa DTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXX_(b)XXXXD XXGH (SEQ ID NO:14), whereinthe initial D is the active site Aspartic acid; the terminal H is theactive site Histidine; X is any amino acid; X_(a) is T, S or F or X_(a)is S or F; and X_(b) is S or R or X_(b) is S; and an amino acid sequencehaving at least 80% amino acid sequence identity to an amino acidsequence of SEQ ID NO:3, 6, or 9, with the proviso that the subtilisinof the BspAP02013-clade of subtilisins and/or recombinant polypeptide oractive fragment thereof of the BspAP02013-clade does not compriseWP_012957833, ERN55058, WP_022626565, or WP_047973355. In still furtherembodiments, a subtilisin of the BspAP02013-clade of subtilisins and/orthe recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises aDTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXX_(b)XXXXDXXGH (SEQ ID NO:14), whereinthe initial D is the active site Aspartic acid; the terminal H is theactive site Histidine; X is any amino acid; X_(a) is S or F; and X_(b)is S; and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9, with theproviso that the subtilisin of the BspAP02013-clade of subtilisinsand/or recombinant polypeptide or active fragment thereof of theBspAP02013-clade does not comprise WP_047973355.

In some embodiments, a subtilisin of the BspAP02013-clade of subtilisinsand/or the recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises a DTGI XXXHXDLXANVXGGXSVFTDSANXDPFXDXXGH (SEQID NO:15) motif, wherein the initial D is the active site Aspartic acidand the terminal H is the active site Histidine, and X is any aminoacid; and an amino acid sequence having at least 80% amino acid sequenceidentity to an amino acid sequence of SEQ ID NO:3, 6, or 9, with theproviso that the subtilisin of the BspAP02013-clade of subtilisinsand/or recombinant polypeptide or active fragment thereof of theBspAP02013-clade does not comprise WP_012957833, ERN55058, orWP_022626565. In some embodiments, a subtilisin of the BspAP02013-cladeof subtilisins and/or the recombinant polypeptide or active fragmentthereof of the BspAP02013-clade comprises a DTGIXXXHXDLXANVXGGXSVFTDSANXDPFXDXXGH (SEQ ID NO:15) motif, wherein the initial Dis the active site Aspartic acid and the terminal H is the active siteHistidine, and X is any amino acid; and an amino acid sequence having atleast 80% amino acid sequence identity to an amino acid sequence of SEQID NO:3, 6, or 9, with the proviso that the subtilisin of theBspAP02013-clade of subtilisins and/or recombinant polypeptide or activefragment thereof of the BspAP02013-clade does not comprise WP_012957833,WP_047973355, ERN55058 or WP_022626565.

In some embodiments, a subtilisin of the BspAP02013-clade of subtilisinsand/or the recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises a DTGI XXXHXDLX_(a)ANVXGGXSVFTDSANXDPFXDXXGH(SEQ ID NO:20) motif, wherein the initial D is the active site Asparticacid; the terminal H is the active site Histidine; X is any amino acid;and X_(a) is S or F; and an amino acid sequence having at least 80%amino acid sequence identity to an amino acid sequence of SEQ ID NO:3,6, or 9, with the proviso that the subtilisin of the BspAP02013-clade ofsubtilisins and/or recombinant polypeptide or active fragment thereof ofthe BspAP02013-clade does not comprise WP_047973355. In someembodiments, a subtilisin of the BspAP02013-clade of subtilisins and/orthe recombinant polypeptide or active fragment thereof of theBspAP02013-clade comprises a DTGIXXXHXDL XANVXGGXSVFTDSANX_(b)DPFXDXXGH(SEQ ID NO:21) motif, wherein the initial D is the active site Asparticacid and the terminal H is the active site Histidine, X is any aminoacid, and X_(b) is S; and an amino acid sequence having at least 80%amino acid sequence identity to an amino acid sequence of SEQ ID NO:3,6, or 9, with the proviso that the subtilisin of the BspAP02013-clade ofsubtilisins and/or recombinant polypeptide or active fragment thereof ofthe BspAP02013-clade does not comprise WP_047973355.

In some embodiments, the BspAP02013-clade of subtilisins comprises asubtilisin or recombinant polypeptide or active fragment thereofcomprising a DTGIXXXHXDLXXXXXGG XSVFXDXXXXXXXXDXXGH (SEQ ID NO:22)motif, wherein the initial D is the active site Aspartic acid and theterminal H is the active site Histidine, and X is any amino acid; and anamino acid sequence having at least 80% amino acid sequence identity toan amino acid sequence of SEQ ID NO:3, 6, or 9, with the proviso thatthe subtilisin of the BspAP02013-clade of subtilisins and/or recombinantpolypeptide or active fragment thereof of the BspAP02013-clade does notcomprise WP_012957833, ERN55058, WP_022626565, or WP_026690432. In otherembodiments, the BspAP02013-clade of subtilisins comprises a subtilisinor recombinant polypeptide or active fragment thereof comprising aDTGIXXXHXDLXXXXXGGXSVFXDX XXXXXXXDXXGH (SEQ ID NO:22) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid; and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9, with the proviso that thesubtilisin of the BspAP02013-clade of subtilisins and/or recombinantpolypeptide or active fragment thereof of the BspAP02013-clade does notcomprise WP_012957833, ERN55058, WP_022626565, WP_026690432, orWP_047973355.

In some embodiments, the BspAP02013-clade of subtilisins comprises asubtilisin or recombinant polypeptide or active fragment thereofcomprising a DTGIXXXHXDLXXNVXGG XSVFXDXXNXDPXXDXXGH (SEQ ID NO:23)motif, wherein the initial D is the active site Aspartic acid and theterminal H is the active site Histidine, and X is any amino acid; and anamino acid sequence having at least 80% amino acid sequence identity toan amino acid sequence of SEQ ID NO:3, 6, or 9, with the proviso thatthe subtilisin of the BspAP02013-clade of subtilisins and/or recombinantpolypeptide or active fragment thereof of the BspAP02013-clade does notcomprise WP_012957833, ERN55058, WP_022626565, or WP_026690432. In someembodiments, the BspAP02013-clade of subtilisins comprises a subtilisinor recombinant polypeptide or active fragment thereof comprising aDTGIXXXHXDLXXNVXGGXSVFXDXX NXDPXXDXXGH (SEQ ID NO:23) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid; and an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or 9, with the proviso that thesubtilisin of the BspAP02013-clade of subtilisins and/or recombinantpolypeptide or active fragment thereof of the BspAP02013-clade does notcomprise WP_012957833, ERN55058, WP_022626565, WP_026690432, orWP_047973355.

In some embodiments, the polypeptide or active fragment thereof of thepresent invention is a polypeptide having a specified degree of aminoacid sequence homology to the exemplified polypeptides, e.g., 70%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the aminoacid sequence selected from the group consisting of SEQ ID NOs: 3, 6,and 9. Some embodiments are directed to a recombinant polypeptide oractive fragment thereof comprising an amino acid sequence having atleast 70% amino acid sequence identity to an amino acid sequence of SEQID NO:3, 6, or 9. Other embodiments are directed to a recombinantpolypeptide or active fragment thereof comprising an amino acid sequencehaving at least 70% amino acid sequence identity to an amino acidsequence of SEQ ID NO:3, 6, or 9, with the proviso that the recombinantpolypeptide or active fragment thereof does not comprise WP_012957833,ERN55058, WP_022626565, WP_026690432, BAD02409, WP_027963976,WP_027965007, WP_026475840, or JP2003325186-0001. Still otherembodiments are directed to a recombinant polypeptide or active fragmentthereof comprising an amino acid sequence having at least 70% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9,with the proviso that the recombinant polypeptide or active fragmentthereof does not comprise WP_047973355, WP_035661169, WP_012957833,ERN55058, WP_022626565, WP_026690432, BAD02409, WP_027963976,WP_027965007, WP_026475840, or JP2003325186-0001. Further embodimentsare directed to a recombinant polypeptide or active fragment thereofcomprising an amino acid sequence having at least 75% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9.Other embodiments are directed to a recombinant polypeptide or activefragment thereof comprising an amino acid sequence having at least 75%amino acid sequence identity to an amino acid sequence of SEQ ID NO:3,6, or 9, with the proviso that the recombinant polypeptide or activefragment thereof does not comprise WP_026690432, WP_012957833, ERN55058,WP_022626565, BAD02409, or JP2003325186-0001. Still other embodimentsare directed to a recombinant polypeptide or active fragment thereofcomprising an amino acid sequence having at least 75% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9,with the proviso that the recombinant polypeptide or active fragmentthereof does not comprise WP_026690432, WP_012957833, ERN55058,WP_022626565, BAD02409, JP2003325186-0001, WP_047973355, orWP_035661169. Even further embodiments are directed to a recombinantpolypeptide or active fragment thereof comprising an amino acid sequencehaving at least 80% amino acid sequence identity to an amino acidsequence of SEQ ID NO:3, 6, or 9. Other embodiments are directed to arecombinant polypeptide or active fragment thereof comprising an aminoacid sequence having at least 80% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9, with the proviso that therecombinant polypeptide or active fragment thereof does not compriseWP_012957833, ERN55058, WP_022626565, or WP_026690432. Still otherembodiments are directed to a recombinant polypeptide or active fragmentthereof comprising an amino acid sequence having at least 80% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9,with the proviso that the recombinant polypeptide or active fragmentthereof does not comprise WP_012957833, ERN55058, WP_022626565,WP_026690432, or WP_047973355. Some embodiments are directed to arecombinant polypeptide or active fragment thereof comprising an aminoacid sequence having at least 85% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9. Other embodiments aredirected to a recombinant polypeptide or active fragment thereofcomprising an amino acid sequence having at least 85% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9,with the proviso that the recombinant polypeptide or active fragmentthereof does not comprise WP_012957833, ERN55058, WP_022626565, orWP_026690432. Other embodiments are directed to a recombinantpolypeptide or active fragment thereof comprising an amino acid sequencehaving at least 85% amino acid sequence identity to an amino acidsequence of SEQ ID NO:3, 6, or 9, with the proviso that the recombinantpolypeptide or active fragment thereof does not comprise WP_012957833,ERN55058, WP_022626565, WP_026690432, or WP_047973355. Other embodimentsare directed to a recombinant polypeptide or active fragment thereofcomprising an amino acid sequence having at least 85% amino acidsequence identity to an amino acid sequence of SEQ ID NO:6 or 9, withthe proviso that the recombinant polypeptide or active fragment thereofdoes not comprise WP_026690432. Some embodiments are directed to arecombinant polypeptide or active fragment thereof comprising an aminoacid sequence having at least 90% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9. Other embodiments aredirected to a recombinant polypeptide or active fragment thereofcomprising an amino acid sequence having at least 90% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9,with the proviso that the recombinant polypeptide or active fragmentthereof does not comprise WP_012957833, ERN55058, or WP_022626565. Otherembodiments are directed to a recombinant polypeptide or active fragmentthereof comprising an amino acid sequence having at least 90% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9,with the proviso that the recombinant polypeptide or active fragmentthereof does not comprise WP_012957833, ERN55058, WP_022626565, orWP_047973355. Other embodiments are directed to a recombinantpolypeptide or active fragment thereof comprising an amino acid sequencehaving at least 90% amino acid sequence identity to an amino acidsequence of SEQ ID NO:6 or 9. Some embodiments are directed to arecombinant polypeptide or active fragment thereof comprising an aminoacid sequence having at least 95% amino acid sequence identity to anamino acid sequence of SEQ ID NO:3, 6, or 9. Other embodiments aredirected to a recombinant polypeptide or active fragment thereofcomprising an amino acid sequence having at least 95% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9,with the proviso that the recombinant polypeptide or active fragmentthereof does not comprise WP_047973355. Still other embodiments aredirected to a recombinant polypeptide or active fragment thereofcomprising an amino acid sequence having at least 97% amino acidsequence identity to an amino acid sequence of SEQ ID NO:3, 6, or 9.Homology can be determined by amino acid sequence alignment, e.g., usinga program such as BLAST, ALIGN, or CLUSTAL, as described herein. In someembodiments, the polypeptide or active fragment thereof is an isolated,recombinant, substantially pure, or non-naturally occurring enzymehaving protease activity, such as subtilisin activity, or caseinhydrolysis activity (for example, dimethylcasein hydrolysis activity).In some embodiments, the polypeptide or active fragment thereof hasprotease activity in the presence of a surfactant.

Also provided is a polypeptide enzyme of the present invention, havingprotease activity, such as alkaline protease activity, said enzymecomprising an amino acid sequence which differs from the amino acidsequence of SEQ ID NO:3, 6 or 9 by no more than 50, no more than 40, nomore than 30, no more than 25, no more than 20, no more than 15, no morethan 10, no more than 9, no more than 8, no more than 7, no more than 6,no more than 5, no more than 4, no more than 3, no more than 2, or nomore than 1 amino acid residue(s), when aligned using any of thepreviously described alignment methods.

As noted above, the variant enzyme polypeptides of the invention haveenzymatic activities (e.g., protease activities) and thus are useful incleaning applications, including but not limited to, methods forcleaning dishware items, tableware items, fabrics, and items having hardsurfaces (e.g., the hard surface of a table, table top, wall, furnitureitem, floor, ceiling, etc.). Exemplary cleaning compositions comprisingone or more variant serine protease enzyme polypeptides of the inventionare described infra. The enzymatic activity (e.g., protease enzymeactivity) of an enzyme polypeptide of the invention can be determinedreadily using procedures well known to those of ordinary skill in theart. The Examples presented infra describe methods for evaluating theenzymatic activity and cleaning performance. The performance ofpolypeptide enzymes of the invention in removing stains (e.g., a proteinstain such as blood/milk/ink or egg yolk), cleaning hard surfaces, orcleaning laundry, dishware or tableware item(s) can be readilydetermined using procedures well known in the art and/or by usingprocedures set forth in the Examples.

The polypeptide or active fragment thereof of the present invention canhave protease activity over a broad range of pH conditions. In someembodiments, the polypeptide or active fragment thereof of the presentinvention have protease activity on dimethylcasein as a substrate, asdemonstrated in Examples below. In some embodiments, the polypeptide oractive fragment thereof of the present invention have protease activityat a pH of from about 4.0 to about 12.0. In some embodiments, thepolypeptide or active fragment thereof of the present invention haveprotease activity at a pH of from about 6.0 to about 12.0. In someembodiments, the polypeptide or active fragment thereof of the presentinvention have at least 50%, 60%, 70%, 80% or 90% of maximal proteaseactivity at a pH of from about 6.0 to about 12.0, or from about 7.0 toabout 12.0. In some embodiments, the polypeptide or active fragmentthereof of the present invention have protease activity at a pH above6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0 or 11.5. Insome embodiments, the polypeptide or active fragment thereof of thepresent invention have protease activity at a pH below 12.0, 11.5, 11.0,10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, or 6.5.

In some embodiments, the polypeptide or active fragment thereof of thepresent invention of the present invention have protease activity at atemperature range from about 10° C. to about 90° C., or from about 30°C. to about 80° C. In some embodiments, the polypeptide or activefragment thereof of the present invention of the present invention haveprotease activity at a temperature range of from about 55° C. to about75° C. In some embodiments, the polypeptide or active fragment thereofof the present invention have at least 50%, 60%, 70%, 80% or 90% ofmaximal protease activity at a temperature of from about 55° C. to about75° C. In some embodiments, the serine proteases have activity at atemperature above 50° C., 55° C., 60° C., 65° C., or 70° C. In someembodiments, the serine proteases have activity at a temperature below75° C., 80° C., 70° C., 65° C., 60° C., or 55° C.

In some embodiments, the polypeptide or active fragment thereof of thepresent invention has at least 50% activity after 20 minutes at 40° C.under stressed conditions. In some embodiments, the polypeptide oractive fragment thereof of the present invention has at least 30%activity after 20 minutes at 50° C. under stressed conditions. In someembodiments, the polypeptide or active fragment thereof of the presentinvention has at least 50% activity after 20 minutes at 40° C. understressed conditions. In some embodiments, the polypeptide or activefragment thereof of the present invention has at least 30% activityafter 20 minutes at 60° C. under stressed conditions. The stressedconditions can be, for example, those shown in Examples. In someembodiments, the stressed condition is in an LAS/EDTA assay, Tris/EDTAassay, or OMO HDL assay.

In some embodiments, the serine protease polypeptides of the presentinvention demonstrate cleaning performance in a cleaning composition.Cleaning compositions often include ingredients harmful to the stabilityand performance of enzymes, making cleaning compositions a harshenvironment for enzymes, e.g. serine proteases, to retain function.Thus, it is not trivial for an enzyme to be put in a cleaningcomposition and expect enzymatic function (e.g. serine proteaseactivity, such as demonstrated by cleaning performance). In someembodiments, the serine protease polypeptides of the present inventiondemonstrate cleaning performance in automatic dishwashing (ADW)detergent compositions. In some embodiments, the cleaning performance inautomatic dishwashing (ADW) detergent compositions includes cleaning ofegg yolk stains. In some embodiments, the serine protease polypeptidesof the present invention demonstrate cleaning performance in laundrydetergent compositions. In some embodiments, the cleaning performance inlaundry detergent compositions includes cleaning of blood/milk/inkstains. In each of the cleaning compositions, the serine proteasepolypeptides of the present invention demonstrate cleaning performancewith or without a bleach component.

A polypeptide of the invention can be subject to various changes, suchas one or more amino acid insertions, deletions, and/or substitutions,either conservative or non-conservative, including where such changes donot substantially alter the enzymatic activity of the polypeptide.Similarly, a nucleic acid of the invention can also be subject tovarious changes, such as one or more substitutions of one or morenucleotides in one or more codons such that a particular codon encodesthe same or a different amino acid, resulting in either a silentvariation (e.g., when the encoded amino acid is not altered by thenucleotide mutation) or non-silent variation, one or more deletions ofone or more nucleic acids (or codons) in the sequence, one or moreadditions or insertions of one or more nucleic acids (or codons) in thesequence, and/or cleavage of or one or more truncations of one or morenucleic acids (or codons) in the sequence. Many such changes in thenucleic acid sequence may not substantially alter the enzymatic activityof the resulting encoded polypeptide enzyme compared to the polypeptideenzyme encoded by the original nucleic acid sequence. A nucleic acidsequence of the invention can also be modified to include one or morecodons that provide for optimum expression in an expression system(e.g., bacterial expression system), while, if desired, said one or morecodons still encode the same amino acid(s).

The invention provides isolated, non-naturally occurring, or recombinantnucleic acids which may be collectively referred to as “nucleic acids ofthe invention” or “polynucleotides of the invention”, which encodepolypeptides of the invention. Nucleic acids of the invention, includingall described below, are useful in recombinant production (e.g.,expression) of polypeptides of the invention, typically throughexpression of a plasmid expression vector comprising a sequence encodingthe polypeptide of interest or fragment thereof. As discussed above,polypeptides include serine protease polypeptides having enzymaticactivity (e.g., proteolytic activity) which are useful in cleaningapplications and cleaning compositions for cleaning an item or a surface(e.g., surface of an item) in need of cleaning.

In some embodiments, a polynucleotide of the present invention is apolynucleotide having a specified degree of nucleic acid homology to theexemplified polynucleotide. In some embodiments, a polynucleotide of thepresent invention has a nucleic acid sequence having at least 50, 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity to SEQ ID NO: 3, 6 or 9. In some embodiments, thepolynucleotide comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1, 4, and 7. In other embodiments, thepolynucleotide of the present invention may also have a complementarynucleic acid sequence to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:1, 4, and 7. Homology can be determined byamino acid sequence alignment, e.g., using a program such as BLAST,ALIGN, or CLUSTAL, as described herein.

In some embodiments, the invention provides an isolated, recombinant,substantially pure, synthetically derived, or non-naturally occurringnucleic acid comprising a nucleotide sequence encoding any polypeptide(including any fusion protein, etc.) of the invention described above inthe section entitled “Polypeptides of the Invention” and elsewhereherein. The invention also provides an isolated, recombinant,substantially pure, synthetically derived or non-naturally-occurringnucleic acid comprising a nucleotide sequence encoding a combination oftwo or more of any polypeptides of the invention described above andelsewhere herein. The present invention provides nucleic acids encodinga serine protease polypeptide of the present invention, wherein theserine protease polypeptide is a mature form having proteolyticactivity. In some embodiments, the serine protease is expressedrecombinantly with a homologous pro-peptide sequence. In otherembodiments, the serine protease is expressed recombinantly with aheterologous pro-peptide sequence (e.g., GG36 pro-peptide sequence).

Nucleic acids of the invention can be generated by using any suitablesynthesis, manipulation, and/or isolation techniques, or combinationsthereof. For example, a polynucleotide of the invention may be producedusing standard nucleic acid synthesis techniques, such as solid-phasesynthesis techniques that are well-known to those skilled in the art. Insuch techniques, fragments of up to 50 or more nucleotide bases aretypically synthesized, then joined (e.g., by enzymatic or chemicalligation methods) to form essentially any desired continuous nucleicacid sequence. The synthesis of the nucleic acids of the invention canbe also facilitated by any suitable method known in the art, includingbut not limited to chemical synthesis using the classicalphosphoramidite method (See e.g., Beaucage et al. Tetrahedron Letters22:1859-69 [1981]); or the method described by Matthes et al. (See,Matthes et al., EMBO J. 3:801-805 [1984], as is typically practiced inautomated synthetic methods. Nucleic acids of the invention also can beproduced by using an automatic DNA synthesizer. Customized nucleic acidscan be ordered from a variety of commercial sources (e.g., The MidlandCertified Reagent Company, the Great American Gene Company, OperonTechnologies Inc., and DNA2.0). Other techniques for synthesizingnucleic acids and related principles are known in the art (See e.g.,Itakura et al., Ann. Rev. Biochem. 53:323 [1984]; and Itakura et al.,Science 198:1056 [1984]).

As indicated above, recombinant DNA techniques useful in modification ofnucleic acids are well known in the art. For example, techniques such asrestriction endonuclease digestion, ligation, reverse transcription andcDNA production, and polymerase chain reaction (e.g., PCR) are known andreadily employed by those of skill in the art. Nucleotides of theinvention may also be obtained by screening cDNA libraries using one ormore oligonucleotide probes that can hybridize to or PCR-amplifypolynucleotides which encode a serine protease polypeptidepolypeptide(s) of the invention. Procedures for screening and isolatingcDNA clones and PCR amplification procedures are well known to those ofskill in the art and described in standard references known to thoseskilled in the art. Some nucleic acids of the invention can be obtainedby altering a naturally occurring polynucleotide backbone (e.g., thatencodes an enzyme or parent protease) by, for example, a knownmutagenesis procedure (e.g., site-directed mutagenesis, site saturationmutagenesis, and in vitro recombination). A variety of methods are knownin the art that are suitable for generating modified polynucleotides ofthe invention that encode serine protease polypeptides of the invention,including, but not limited to, for example, site-saturation mutagenesis,scanning mutagenesis, insertional mutagenesis, deletion mutagenesis,random mutagenesis, site-directed mutagenesis, and directed-evolution,as well as various other recombinatorial approaches.

The present invention provides vectors comprising at least one serineprotease polynucleotide of the invention described herein (e.g., apolynucleotide encoding a serine protease polypeptide of the inventiondescribed herein), expression vectors or expression cassettes comprisingat least one nucleic acid or polynucleotide of the invention, isolated,substantially pure, or recombinant DNA constructs comprising at leastone nucleic acid or polynucleotide of the invention, isolated orrecombinant cells comprising at least one polynucleotide of theinvention, and compositions comprising one or more such vectors, nucleicacids, expression vectors, expression cassettes, DNA constructs, cells,cell cultures, or any combination or mixtures thereof.

In some embodiments, the invention provides recombinant cells comprisingat least one vector (e.g., expression vector or DNA construct) of theinvention which comprises at least one nucleic acid or polynucleotide ofthe invention. Some such recombinant cells are transformed ortransfected with such at least one vector, although other methods areavailable and known in the art. Such cells are typically referred to ashost cells. Some such cells comprise bacterial cells, including, but arenot limited to Bacillus sp. cells, such as B. subtilis cells. Theinvention also provides recombinant cells (e.g., recombinant host cells)comprising at least one serine protease polypeptide of the invention.

In some embodiments, the invention provides a vector comprising anucleic acid or polynucleotide of the invention. In some embodiments,the vector is an expression vector or expression cassette in which apolynucleotide sequence of the invention which encodes a serine proteasepolypeptide of the invention is operably linked to one or additionalnucleic acid segments required for efficient gene expression (e.g., apromoter operably linked to the polynucleotide of the invention whichencodes a serine protease polypeptide of the invention). A vector mayinclude a transcription terminator and/or a selection gene, such as anantibiotic resistance gene, that enables continuous cultural maintenanceof plasmid-infected host cells by growth in antimicrobial-containingmedia.

An expression vector may be derived from plasmid or viral DNA, or inalternative embodiments, contains elements of both. Exemplary vectorsinclude, but are not limited to pC194, pJH101, pE194, pHP13 (See,Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods forBacillus, John Wiley & Sons [1990]; suitable replicating plasmids for B.subtilis include those listed on p. 92) See also, Perego, IntegrationalVectors for Genetic Manipulations in Bacillus subtilis, in Sonenshein etal., [eds.] Bacillus subtilis and Other Gram-Positive Bacteria:Biochemistry, Physiology and Molecular Genetics, American Society forMicrobiology, Washington, D.C. [1993], pp. 615-624), and p2JM103BBI.

For expression and production of a protein of interest (e.g., serineprotease polypeptide) in a cell, at least one expression vectorcomprising at least one copy of a polynucleotide encoding the serineprotease polypeptide, and in some instances comprising multiple copies,is transformed into the cell under conditions suitable for expression ofthe serine protease. In some embodiments of the present invention, apolynucleotide sequence encoding the serine protease polypeptide (aswell as other sequences included in the vector) is integrated into thegenome of the host cell, while in other embodiments, a plasmid vectorcomprising a polynucleotide sequence encoding the serine proteasepolypeptide remains as autonomous extrachromosomal element within thecell. The invention provides both extrachromosomal nucleic acid elementsas well as incoming nucleotide sequences that are integrated into thehost cell genome. The vectors described herein are useful for productionof the serine protease polypeptides of the invention. In someembodiments, a polynucleotide construct encoding the serine proteasepolypeptide is present on an integrating vector that enables theintegration and optionally the amplification of the polynucleotideencoding the serine protease polypeptide into the host chromosome.Examples of sites for integration are well known to those skilled in theart. In some embodiments, transcription of a polynucleotide encoding aserine protease polypeptide of the invention is effectuated by apromoter that is the wild-type promoter for the selected precursorprotease. In some other embodiments, the promoter is heterologous to theprecursor protease, but is functional in the host cell. Specifically,examples of suitable promoters for use in bacterial host cells include,but are not limited to, for example, the amyE, amyQ, amyL, pstS, sacB,pSPAC, pAprE, pVeg, pHpaII promoters, the promoter of the B.stearothermophilus maltogenic amylase gene, the B. amyloliquefaciens(BAN) amylase gene, the B. subtilis alkaline protease gene, the B.clausii alkaline protease gene the B. pumilis xylosidase gene, the B.thuringiensis cryIIIA, and the B. licheniformis alpha-amylase gene.Additional promoters include, but are not limited to the A4 promoter, aswell as phage Lambda PR or PL promoters, and the E. coli lac, trp or tacpromoters.

Serine protease polypeptides of the present invention can be produced inhost cells of any suitable microorganism, including bacteria and fungi.In some embodiments, serine protease polypeptides of the presentinvention can be produced in Gram-positive bacteria. In someembodiments, the host cells are Bacillus spp., Streptomyces spp.,Escherichia spp., Aspergillus spp., Trichoderma spp., Pseudomonas spp.,Corynebacterium spp., Saccharomyces spp., or Pichia spp. In someembodiments, the serine protease polypeptides are produced by Bacillussp. host cells. Examples of Bacillus sp. host cells that find use in theproduction of the serine protease polypeptides of the invention include,but are not limited to B. licheniformis, B. lentus, B. subtilis, B.amyloliquefaciens, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. coagulans, B. circulans, B. pumilis, B. thuringiensis,B. clausii, and B. megaterium, as well as other organisms within thegenus Bacillus. In some embodiments, B. subtilis host cells are used forproduction of serine protease polypeptides. U.S. Pat. Nos. 5,264,366 and4,760,025 (RE 34,606) describe various Bacillus host strains that can beused for producing serine protease polypeptide of the invention,although other suitable strains can be used.

Several bacterial strains that can be used to produce serine proteasepolypeptides of the invention include non-recombinant (i.e., wild-type)Bacillus sp. strains, as well as variants of naturally-occurring strainsand/or recombinant strains. In some embodiments, the host strain is arecombinant strain, wherein a polynucleotide encoding a polypeptide ofinterest has been introduced into the host. In some embodiments, thehost strain is a B. subtilis host strain and particularly a recombinantB. subtilis host strain. Numerous B. subtilis strains are known,including, but not limited to, for example, 1A6 (ATCC 39085), 168(1A01), SB19, W23, Ts85, B637, PB1753 through PB1758, PB3360, JH642,1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094),GX4931, PBT 110, and PEP 211strain (See e.g., Hoch et al., Genetics73:215-228 [1973]; See also, U.S. Pat. Nos. 4,450,235 and 4,302,544, andEP0134048, each of which is incorporated by reference in its entirety).The use of B. subtilis as an expression host cells is well known in theart (See e.g., Palva et al., Gene 19:81-87 [1982]; Fahnestock andFischer, J. Bacteriol., 165:796-804 [1986]; and Wang et al., Gene69:39-47 [1988]).

In some embodiments, the Bacillus host cell is a Bacillus sp. thatincludes a mutation or deletion in at least one of the following genes,degU, degS, degR and degQ. In some embodiments, the mutation is in adegU gene, and in some embodiments the mutation is degU(Hy)32 (See e.g.,Msadek et al., J. Bacteriol. 172:824-834 [1990]; and Olmos et al., Mol.Gen. Genet. 253:562-567 [1997]). In some embodiments, the Bacillus hostcomprises a mutation or deletion in scoC4 (See e.g., Caldwell et al., J.Bacteriol. 183:7329-7340 [2001]); spoIIE (See e.g., Arigoni et al., Mol.Microbiol. 31:1407-1415 [1999]); and/or oppA or other genes of the oppoperon (See e.g., Perego et al., Mol. Microbiol. 5:173-185 [1991]).Indeed, it is contemplated that any mutation in the opp operon thatcauses the same phenotype as a mutation in the oppA gene will find usein some embodiments of the altered Bacillus strain of the invention. Insome embodiments, these mutations occur alone, while in otherembodiments, combinations of mutations are present. In some embodiments,an altered Bacillus host cell strain that can be used to produce aserine protease polypeptide of the invention is a Bacillus host strainthat already includes a mutation in one or more of the above-mentionedgenes. In addition, Bacillus sp. host cells that comprise mutation(s)and/or deletions of endogenous protease genes find use. In someembodiments, the Bacillus host cell comprises a deletion of the aprE andthe nprE genes. In other embodiments, the Bacillus sp. host cellcomprises a deletion of 5 protease genes, while in other embodiments,the Bacillus sp. host cell comprises a deletion of 9 protease genes (Seee.g., US2005/0202535, incorporated herein by reference).

Host cells are transformed with at least one nucleic acid encoding atleast one serine protease polypeptide of the invention using anysuitable method known in the art. Methods for introducing a nucleic acid(e.g., DNA) into Bacillus cells or E. coli cells utilizing plasmid DNAconstructs or vectors and transforming such plasmid DNA constructs orvectors into such cells are well known. In some embodiments, theplasmids are subsequently isolated from E. coli cells and transformedinto Bacillus cells. However, it is not essential to use interveningmicroorganisms such as E. coli, and in some embodiments, a DNA constructor vector is directly introduced into a Bacillus host.

Those of skill in the art are well aware of suitable methods forintroducing nucleic acid sequences of the invention into Bacillus cells(See e.g., Ferrari et al., “Genetics,” in Harwood et al. [eds.],Bacillus, Plenum Publishing Corp. [1989], pp. 57-72; Saunders et al., J.Bacteriol. 157:718-726 [1984]; Hoch et al., J. Bacteriol. 93:1925-1937[1967]; Mann et al., Current Microbiol. 13:131-135 [1986]; Holubova,Folia Microbiol. 30:97 [1985]; Chang et al., Mol. Gen. Genet. 168:11-115[1979]; Vorobjeva et al., FEMS Microbiol. Lett. 7:261-263 [1980]; Smithet al., Appl. Env. Microbiol. 51:634 [1986]; Fisher et al., Arch.Microbiol. 139:213-217 [1981]; and McDonald, J. Gen. Microbiol. 130:203[1984]). Indeed, such methods as transformation, including protoplasttransformation and transfection, transduction, and protoplast fusion arewell known and suited for use in the present invention. Methods known inthe art to transform Bacillus cells include such methods as plasmidmarker rescue transformation, which involves the uptake of a donorplasmid by competent cells carrying a partially homologous residentplasmid (See, Contente et al., Plasmid 2:555-571 [1979]; Haima et al.,Mol. Gen. Genet. 223:185-191 [1990]; Weinrauch et al., J. Bacteriol.154:1077-1087 [1983]; and Weinrauch et al., J. Bacteriol. 169:1205-1211[1987]). In this method, the incoming donor plasmid recombines with thehomologous region of the resident “helper” plasmid in a process thatmimics chromosomal transformation.

In addition to commonly used methods, in some embodiments, host cellsare directly transformed with a DNA construct or vector comprising anucleic acid encoding a serine protease polypeptide of the invention(i.e., an intermediate cell is not used to amplify, or otherwiseprocess, the DNA construct or vector prior to introduction into the hostcell). Introduction of the DNA construct or vector of the invention intothe host cell includes those physical and chemical methods known in theart to introduce a nucleic acid sequence (e.g., DNA sequence) into ahost cell without insertion into the host genome. Such methods include,but are not limited to calcium chloride precipitation, electroporation,naked DNA, liposomes and the like. In additional embodiments, DNAconstructs or vector are co-transformed with a plasmid, without beinginserted into the plasmid. In further embodiments, a selective marker isdeleted from the altered Bacillus strain by methods known in the art(See, Stahl et al., J. Bacteriol. 158:411-418 [1984]; and Palmeros etal., Gene 247:255-264 [2000]).

In some embodiments, the transformed cells of the present invention arecultured in conventional nutrient media. The suitable specific cultureconditions, such as temperature, pH and the like are known to thoseskilled in the art and are well described in the scientific literature.In some embodiments, the invention provides a culture (e.g., cellculture) comprising at least one serine protease polypeptide or at leastone nucleic acid of the invention.

In some embodiments, host cells transformed with at least onepolynucleotide sequence encoding at least one serine proteasepolypeptide of the invention are cultured in a suitable nutrient mediumunder conditions permitting the expression of the present protease,after which the resulting protease is recovered from the culture. Insome embodiments, the protease produced by the cells is recovered fromthe culture medium by conventional procedures, including, but notlimited to for example, separating the host cells from the medium bycentrifugation or filtration, precipitating the proteinaceous componentsof the supernatant or filtrate by means of a salt (e.g., ammoniumsulfate), chromatographic purification (e.g., ion exchange, gelfiltration, affinity, etc.).

In some embodiments, a serine protease polypeptide produced by arecombinant host cell is secreted into the culture medium. A nucleicacid sequence that encodes a purification facilitating domain may beused to facilitate purification of proteins. A vector or DNA constructcomprising a polynucleotide sequence encoding a serine proteasepolypeptide may further comprise a nucleic acid sequence encoding apurification facilitating domain to facilitate purification of theserine protease polypeptide (See e.g., Kroll et al., DNA Cell Biol.12:441-53 [1993]). Such purification facilitating domains include, butare not limited to, for example, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals (See, Porath, Protein Expr. Purif. 3:263-281 [1992]), protein Adomains that allow purification on immobilized immunoglobulin, and thedomain utilized in the FLAGS extension/affinity purification system. Theinclusion of a cleavable linker sequence such as Factor XA orenterokinase (e.g., sequences available from Invitrogen, San Diego,Calif.) between the purification domain and the heterologous proteinalso find use to facilitate purification.

Assays for detecting and measuring the enzymatic activity of an enzyme,such as a serine protease polypeptide of the invention, are well known.Various assays for detecting and measuring activity of proteases (e.g.,serine protease polypeptides of the invention), are also known to thoseof ordinary skill in the art. In particular, assays are available formeasuring protease activity that are based on the release ofacid-soluble peptides from casein or hemoglobin, measured as absorbanceat 280 nm or colorimetrically using the Folin method. Other exemplaryassays involve the solubilization of chromogenic substrates (See e.g.,Ward, “Proteinases,” in Fogarty (ed.)., Microbial Enzymes andBiotechnology, Applied Science, London, [1983], pp. 251-317). Otherexemplary assays include, but are not limited tosuccinyl-Ala-Ala-Pro-Phe-para nitroanilide assay (suc-AAPF-pNA) and the2,4,6-trinitrobenzene sulfonate sodium salt assay (TNBS assay). Numerousadditional references known to those in the art provide suitable methods(See e.g., Wells et al., Nucleic Acids Res. 11:7911-7925 [1983];Christianson et al., Anal. Biochem. 223:119-129 [1994]; and Hsia et al.,Anal Biochem. 242:221-227 [1999]).

A variety of methods can be used to determine the level of production ofa mature protease (e.g., mature serine protease polypeptides of thepresent invention) in a host cell. Such methods include, but are notlimited to, for example, methods that utilize either polyclonal ormonoclonal antibodies specific for the protease. Exemplary methodsinclude, but are not limited to enzyme-linked immunosorbent assays(ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), andfluorescent activated cell sorting (FACS). These and other assays arewell known in the art (See e.g., Maddox et al., J. Exp. Med. 158:1211[1983]).

In some other embodiments, the invention provides methods for making orproducing a mature serine protease polypeptide of the invention. Amature serine protease polypeptide does not include a signal peptide ora propeptide sequence. Some methods comprise making or producing aserine protease polypeptide of the invention in a recombinant bacterialhost cell, such as for example, a Bacillus sp. cell (e.g., a B. subtiliscell). In some embodiments, the invention provides a method of producinga serine protease polypeptide of the invention, the method comprisingcultivating a recombinant host cell comprising a recombinant expressionvector comprising a nucleic acid encoding a serine protease polypeptideof the invention under conditions conducive to the production of theserine protease polypeptide. Some such methods further compriserecovering the serine protease polypeptide from the culture.

In some embodiments the invention provides methods of producing a serineprotease polypeptide of the invention, the methods comprising: (a)introducing a recombinant expression vector comprising a nucleic acidencoding a serine protease polypeptide of the invention into apopulation of cells (e.g., bacterial cells, such as B. subtilis cells);and (b) culturing the cells in a culture medium under conditionsconducive to produce the serine protease polypeptide encoded by theexpression vector. Some such methods further comprise: (c) isolating theserine protease polypeptide from the cells or from the culture medium.

Unless otherwise noted, all component or composition levels providedherein are made in reference to the active level of that component orcomposition, and are exclusive of impurities, for example, residualsolvents or by-products, which may be present in commercially availablesources. Enzyme components weights are based on total active protein.All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated. Compositions of the inventioninclude cleaning compositions, such as detergent compositions. In theexemplified detergent compositions, the enzymes levels are expressed bypure enzyme by weight of the total composition and unless otherwisespecified, the detergent ingredients are expressed by weight of thetotal compositions.

While not essential for the purposes of the present invention, thenon-limiting list of adjuncts illustrated hereinafter are suitable foruse in the instant cleaning compositions. In some embodiments, theseadjuncts are incorporated for example, to assist or enhance cleaningperformance, for treatment of the substrate to be cleaned, or to modifythe aesthetics of the cleaning composition as is the case with perfumes,colorants, dyes or the like. It is understood that such adjuncts are inaddition to the serine protease polypeptides of the present invention.The precise nature of these additional components, and levels ofincorporation thereof, will depend on the physical form of thecomposition and the nature of the cleaning operation for which it is tobe used. Suitable adjunct materials include, but are not limited to,bleach catalysts, other enzymes, enzyme stabilizing systems, chelants,optical brighteners, soil release polymers, dye transfer agents,dispersants, suds suppressors, dyes, perfumes, colorants, filler salts,photoactivators, fluorescers, fabric conditioners, hydrolyzablesurfactants, preservatives, antioxidants, anti-shrinkage agents,anti-wrinkle agents, germicides, fungicides, color speckles, silvercare,anti-tarnish and/or anti-corrosion agents, alkalinity sources,solubilizing agents, carriers, processing aids, pigments, and pH controlagents, surfactants, builders, chelating agents, dye transfer inhibitingagents, deposition aids, dispersants, additional enzymes, and enzymestabilizers, catalytic materials, bleach activators, bleach boosters,hydrogen peroxide, sources of hydrogen peroxide, preformed peracids,polymeric dispersing agents, clay soil removal/anti-redeposition agents,brighteners, suds suppressors, dyes, perfumes, structure elasticizingagents, fabric softeners, carriers, hydrotropes, processing aids and/orpigments. In addition to the disclosure below, suitable examples of suchother adjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282;6,306,812; 6,326,348; 6,610,642; 6,605,458; 5,705,464; 5,710,115;5,698,504; 5,695,679; 5,686,014; and 5,646,101, all of which areincorporated herein by reference. In embodiments in which the cleaningadjunct materials are not compatible with the serine proteasepolypeptides of the present invention in the cleaning compositions, thensuitable methods of keeping the cleaning adjunct materials and theprotease(s) separated (i.e., not in contact with each other) untilcombination of the two components is appropriate are used. Suchseparation methods include any suitable method known in the art (e.g.,gelcaps, encapsulation, tablets, physical separation, etc.). Theaforementioned adjunct ingredients may constitute the balance of thecleaning compositions of the present invention.

The cleaning compositions of the present invention are advantageouslyemployed for example, in laundry applications, hard surface cleaningapplications, dishwashing applications, including automatic dishwashingand hand dishwashing, as well as cosmetic applications such as dentures,teeth, hair and skin cleaning. The enzymes of the present invention arealso suited for use in contact lens cleaning and wound debridementapplications. In addition, due to the unique advantages of increasedeffectiveness in lower temperature solutions, the enzymes of the presentinvention are ideally suited for laundry applications. Furthermore, theenzymes of the present invention find use in granular and liquidcompositions.

The serine protease polypeptides of the present invention also find usein cleaning additive products. In some embodiments, low temperaturesolution cleaning applications find use. In some embodiments, thepresent invention provides cleaning additive products including at leastone enzyme of the present invention is ideally suited for inclusion in awash process when additional bleaching effectiveness is desired. Suchinstances include, but are not limited to low temperature solutioncleaning applications. In some embodiments, the additive product is inits simplest form, one or more proteases. In some embodiments, theadditive is packaged in dosage form for addition to a cleaning process.In some embodiments, the additive is packaged in dosage form foraddition to a cleaning process where a source of peroxygen is employedand increased bleaching effectiveness is desired. Any suitable singledosage unit form finds use with the present invention, including but notlimited to pills, tablets, gelcaps, or other single dosage units such aspre-measured powders or liquids. In some embodiments, filler(s) orcarrier material(s) are included to increase the volume of suchcompositions. Suitable filler or carrier materials include, but are notlimited to, various salts of sulfate, carbonate and silicate as well astalc, clay and the like. Suitable filler or carrier materials for liquidcompositions include, but are not limited to water or low molecularweight primary and secondary alcohols including polyols and diols.Examples of such alcohols include, but are not limited to, methanol,ethanol, propanol and isopropanol. In some embodiments, the compositionscontain from about 5 to about 90% of such materials. Acidic fillers finduse to reduce pH. Alternatively, in some embodiments, the cleaningadditive includes adjunct ingredients, as more fully described below.

The present cleaning compositions and cleaning additives require aneffective amount of at least one of the serine protease polypeptidesprovided herein, alone or in combination with other proteases and/oradditional enzymes. The required level of enzyme is achieved by theaddition of one or more serine protease polypeptides of the presentinvention. Typically the present cleaning compositions comprise at leastabout 0.0001 weight percent, from about 0.0001 to about 10, from about0.001 to about 1, or from about 0.01 to about 0.1 weight percent of atleast one of the serine protease polypeptides of the present invention.

The cleaning compositions herein are typically formulated such that,during use in aqueous cleaning operations, the wash water will have a pHof from about 4.0 to about 11.5, or even from about 5.0 to about 11.5,or even from about 5.0 to about 8.0, or even from about 7.5 to about10.5. Liquid product formulations are typically formulated to have a pHfrom about 3.0 to about 9.0 or even from about 3 to about 5. Granularlaundry products are typically formulated to have a pH from about 9 toabout 11. In some embodiments, the cleaning compositions of the presentinvention can be formulated to have an alkaline pH under washconditions, such as a pH of from about 8.0 to about 12.0, or from about8.5 to about 11.0, or from about 9.0 to about 11.0. In some embodiments,the cleaning compositions of the present invention can be formulated tohave a neutral pH under wash conditions, such as a pH of from about 5.0to about 8.0, or from about 5.5 to about 8.0, or from about 6.0 to about8.0, or from about 6.0 to about 7.5. In some embodiments, the neutral pHconditions can be measured when the cleaning composition is dissolved1:100 (wt:wt) in de-ionised water at 20° C., measured using aconventional pH meter. Techniques for controlling pH at recommendedusage levels include the use of buffers, alkalis, acids, etc., and arewell known to those skilled in the art.

In some embodiments, when the serine protease polypeptide (s) is/areemployed in a granular composition or liquid, it is desirable for theserine protease polypeptide to be in the form of an encapsulatedparticle to protect the serine protease polypeptide from othercomponents of the granular composition during storage. In addition,encapsulation is also a means of controlling the availability of theserine protease polypeptide during the cleaning process. In someembodiments, encapsulation enhances the performance of the serineprotease polypeptide (s) and/or additional enzymes. In this regard, theserine protease polypeptides of the present invention are encapsulatedwith any suitable encapsulating material known in the art. In someembodiments, the encapsulating material typically encapsulates at leastpart of the serine protease polypeptide (s) of the present invention.Typically, the encapsulating material is water-soluble and/orwater-dispersible. In some embodiments, the encapsulating material has aglass transition temperature (Tg) of 0° C. or higher. Glass transitiontemperature is described in more detail in WO97/11151. The encapsulatingmaterial is typically selected from consisting of carbohydrates, naturalor synthetic gums, chitin, chitosan, cellulose and cellulosederivatives, silicates, phosphates, borates, polyvinyl alcohol,polyethylene glycol, paraffin waxes, and combinations thereof. When theencapsulating material is a carbohydrate, it is typically selected frommonosaccharides, oligosaccharides, polysaccharides, and combinationsthereof. In some typical embodiments, the encapsulating material is astarch (See e.g., EP0922499; U.S. Pat. Nos. 4,977,252; 5,354,559, and5,935,826). In some embodiments, the encapsulating material is amicrosphere made from plastic such as thermoplastics, acrylonitrile,methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixturesthereof; commercially available microspheres that find use include, butare not limited to those supplied by EXPANCEL® (Stockviksverken,Sweden); and PM6545, PM6550, PM7220, PM7228, EXTENDOSPHERES®, LUXSIL®,Q-CEL®, and SPHERICEL® (PQ Corp., Valley Forge, Pa.).

There are a variety of wash conditions including varying detergentformulations, wash water volumes, wash water temperatures, and lengthsof wash time, to which proteases involved in washing are exposed. A lowdetergent concentration system includes detergents where less than about800 ppm of the detergent components are present in the wash water. Amedium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of the detergent components are present inthe wash water. A high detergent concentration system includesdetergents where greater than about 2000 ppm of the detergent componentsare present in the wash water. In some embodiments, the “cold waterwashing” of the present invention utilizes “cold water detergent”suitable for washing at temperatures from about 10° C. to about 40° C.,or from about 20° C. to about 30° C., or from about 15° C. to about 25°C., as well as all other combinations within the range of about 15° C.to about 35° C., and all ranges within 10° C. to 40° C.

Different geographies typically have different water hardness. Waterhardness is usually described in terms of the grains per gallon mixedCa²⁺/Mg²⁺. Hardness is a measure of the amount of calcium (Ca²⁺) andmagnesium (Mg²⁺) in the water. Most water in the United States is hard,but the degree of hardness varies. Moderately hard (60-120 ppm) to hard(121-181 ppm) water has 60 to 181 parts per million.

TABLE I Water Hardness Water Grains per gallon Parts per million Softless than 1.0 less than 17 Slightly hard 1.0 to 3.5 17 to 60 Moderatelyhard 3.5 to 7.0 60 to 120 Hard 7.0 to 10.5 120 to 180 Very hard greaterthan 10.5 greater than 180

Accordingly, in some embodiments, the present invention provides serineprotease polypeptides that show surprising wash performance in at leastone set of wash conditions (e.g., water temperature, water hardness,and/or detergent concentration). In some embodiments, the serineprotease polypeptides of the present invention are comparable in washperformance to other serine protease polypeptide proteases. In someembodiments of the present invention, the serine protease polypeptidesprovided herein exhibit enhanced oxidative stability, enhanced thermalstability, enhanced cleaning capabilities under various conditions,and/or enhanced chelator stability. In addition, the serine proteasepolypeptides of the present invention find use in cleaning compositionsthat do not include detergents, again either alone or in combinationwith builders and stabilizers.

In some embodiments of the present invention, the cleaning compositionscomprise at least one serine protease polypeptide of the presentinvention at a level from about 0.00001 to about 10% by weight of thecomposition and the balance (e.g., about 99.999 to about 90.0%)comprising cleaning adjunct materials by weight of composition. In someother embodiments of the present invention, the cleaning compositions ofthe present invention comprises at least one serine protease polypeptideat a level of about 0.0001 to about 10%, about 0.001 to about 5%, about0.001 to about 2%, about 0.005 to about 0.5% by weight of thecomposition and the balance of the cleaning composition (e.g., about99.9999 to about 90.0%, about 99.999 to about 98%, about 99.995 to about99.5% by weight) comprising cleaning adjunct materials.

In some embodiments, the cleaning compositions of the present inventioncomprise one or more additional detergent enzymes, which providecleaning performance and/or fabric care and/or dishwashing benefits.Examples of suitable enzymes include, but are not limited to, acyltransferases, alpha-amylases, beta-amylases, alpha-galactosidases,arabinosidases, aryl esterases, beta-galactosidases, carrageenases,catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases,endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases,exo-mannanases, galactanases, glucoamylases, hemicellulases,hyaluronidases, keratinases, laccases, lactases, ligninases, lipases,lipoxygenases, mannanases, oxidases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, peroxidases, phenoloxidases,phosphatases, phospholipases, phytases, polygalacturonases, proteases,pullulanases, reductases, rhamnogalacturonases, beta-glucanases,tannases, transglutaminases, xylan acetyl-esterases, xylanases,xyloglucanases, and xylosidases, or any combinations or mixturesthereof. In some embodiments, a combination of enzymes is used (i.e., a“cocktail”) comprising conventional applicable enzymes like protease,lipase, cutinase and/or cellulase in conjunction with amylase is used.

In addition to the serine protease polypeptides provided herein, anyother suitable protease finds use in the compositions of the presentinvention. Suitable proteases include those of animal, vegetable ormicrobial origin. In some embodiments, microbial proteases are used. Insome embodiments, chemically or genetically modified mutants areincluded. In some embodiments, the protease is a serine protease,preferably an alkaline microbial protease or a trypsin-like protease.Examples of alkaline proteases include subtilisins, especially thosederived from Bacillus (e.g., subtilisin, lentus, amyloliquefaciens,subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin168). Additional examples include those mutant proteases described in USRE 34,606; 5,955,340; 5,700,676; 6,312,936; and 6,482,628, all of whichare incorporated herein by reference. Additional protease examplesinclude, but are not limited to, trypsin (e.g., of porcine or bovineorigin), and the Fusarium protease described in WO89/06270. In someembodiments, commercially available protease enzymes that find use inthe present invention include, but are not limited to MAXATASE®,MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®,PURAFECT® OXP, PURAlVIAX™, EXCELLASE™, PREFERENZ™ proteases (e.g. P100,P110, P280), EFFECTENZ™ proteases (e.g. P1000, P1050, P2000), EXCELLENZ™proteases (e.g. P1000), ULTIMASE®, and PURAFAST™ (Genencor); ALCALASE®,SAVINASE®, PRIMASE®, DURAZYM™, POLARZYME®, OVOZYME®, KANNASE®,LIQUANASE®, NEUTRASE®, RELASE® and ESPERASE® (Novozymes); BLAP™ andBLAP™ (Henkel Kommanditgesellschaft auf Aktien, Duesseldorf, Germany),and KAP (B. alkalophilus subtilisin; Kao Corp., Tokyo, Japan). Variousproteases are described in WO95/23221, WO 92/21760, WO09/149200,WO09/149144, WO09/149145, WO11/072099, WO10/056640, WO 10/056653,WO11/140364, WO12/151534, US 2008/0090747; 5,801,039; 5,340,735;5,500,364; 5,855,625; RE 34,606; 5,955,340; 5,700,676; 6,312,936;6,482,628; 8,530,219; and various other patents. In some furtherembodiments, neutral metalloproteases find use in the present invention,including but not limited to the neutral metalloproteases described inWO1999014341, WO 1999033960, WO1999014342, WO1999034003, WO2007044993,WO2009058303, WO 2009058661, WO2014/071410, WO2014/194032,WO2014/194034, WO2014/194054, and WO2014/194117. Exemplarymetalloproteases include nprE, the recombinant form of neutralmetalloprotease expressed in B. subtilis (See e.g., WO07/044993), andPMN, the purified neutral metalloprotease from B. amyloliquefaciens.

In addition, any suitable lipase finds use in the present invention.Suitable lipases include, but are not limited to those of bacterial orfungal origin. Chemically or genetically modified mutants areencompassed by the present invention. Examples of useful lipases includeH. lanuginosa lipase (See e.g., EP258068, and EP305216), Rhizomucormiehei lipase (See e.g., EP238023), Candida lipase, such as C.antarctica lipase (e.g., the C. antarctica lipase A or B; See e.g.,EP214761), Pseudomonas lipases such as P. alcaligenes lipase and P.pseudoalcaligenes lipase (See e.g., EP218 272), P. cepacia lipase (Seee.g., EP331376), P. stutzeri lipase (See e.g., GB1,372,034), P.fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase [Dartoiset al., Biochem. Biophys. Acta 1131:253-260 [1993]); B.stearothermophilus lipase [See e.g., JP 64/744992]; and B. pumiluslipase [See e.g., WO91/16422]).

Furthermore, a number of cloned lipases find use in some embodiments ofthe present invention, including but not limited to Penicilliumcamembertii lipase (See, Yamaguchi et al., Gene 103:61-67 [1991]),Geotricum candidum lipase (See, Schimada et al., J. Biochem.,106:383-388 [1989]), and various Rhizopus lipases such as R. delemarlipase (See, Hass et al., Gene 109:117-113 [1991]), a R. niveus lipase(Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 [1992]) and R.oryzae lipase.

Other types of lipase polypeptide enzymes such as cutinases also finduse in some embodiments of the present invention, including but notlimited to the cutinase derived from Pseudomonas mendocina (See,WO88/09367), and the cutinase derived from Fusarium solani pisi (See,WO90/09446).

Additional suitable lipases include lipases such as M1 LIPASE™, LUMAFAST™, and LIPOMAX™ (Genencor); LIPEX®, LIPOLASE® and LIPOLASE® ULTRA(Novozymes); and LIPASE P™ “Amano” (Amano Pharmaceutical Co. Ltd.,Japan).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise lipases at a level from about0.00001 to about 10% of additional lipase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In some other embodiments of the present invention, the cleaningcompositions of the present invention also comprise lipases at a levelof about 0.0001 to about 10%, about 0.001 to about 5%, about 0.001 toabout 2%, about 0.005 to about 0.5% lipase by weight of the composition.

In some embodiments of the present invention, any suitable amylase findsuse in the present invention. In some embodiments, any amylase (e.g.,alpha and/or beta) suitable for use in alkaline solutions also find use.Suitable amylases include, but are not limited to those of bacterial orfungal origin. Chemically or genetically modified mutants are includedin some embodiments. Amylases that find use in the present invention,include, but are not limited to α-amylases obtained from B.licheniformis (See e.g., GB 1,296,839). Additional suitable amylasesinclude those found in WO9510603, WO9526397, WO9623874, WO9623873,WO9741213, WO 9919467, WO0060060, WO0029560, WO9923211, WO9946399,WO0060058, WO0060059, WO9942567, WO0114532, WO02092797, WO0166712,WO0188107, WO0196537, WO 0210355, WO9402597, WO0231124, WO9943793,WO9943794, WO2004113551, WO 2005001064, WO2005003311, WO0164852,WO2006063594, WO2006066594, WO 2006066596, WO2006012899, WO2008092919,WO2008000825, WO2005018336, WO 2005066338, WO2009140504, WO2005019443,WO2010091221, WO2010088447, WO 0134784, WO2006012902, WO2006031554,WO2006136161, WO2008101894, WO 2010059413, WO2011098531, WO2011080352,WO2011080353, WO2011080354, WO 2011082425, WO2011082429, WO2011076123,WO2011087836, WO2011076897, WO 94183314, WO9535382, WO9909183,WO9826078, WO9902702, WO9743424, WO9929876, WO9100353, WO9605295,WO9630481, WO9710342, WO2008088493, WO2009149419, WO 2009061381,WO2009100102, WO2010104675, WO2010117511, and WO2010115021. Commerciallyavailable amylases that find use in the present invention include, butare not limited to DURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYMEPLUS®, STAINZYME ULTRA®, and BAN™ (Novozymes), as well as POWERASE™RAPIDASE® and MAXAMYL® P (Genencor).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise amylases at a level from about0.00001 to about 10% of additional amylase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In some other embodiments of the present invention, the cleaningcompositions of the present invention also comprise amylases at a levelof about 0.0001 to about 10%, about 0.001 to about 5%, about 0.001 toabout 2%, about 0.005 to about 0.5% amylase by weight of thecomposition.

In some further embodiments, any suitable cellulase finds used in thecleaning compositions of the present invention. Suitable cellulasesinclude, but are not limited to those of bacterial or fungal origin.Chemically or genetically modified mutants are included in someembodiments. Suitable cellulases include, but are not limited to H.insolens cellulases (See e.g., U.S. Pat. No. 4,435,307). Especiallysuitable cellulases are the cellulases having color care benefits (Seee.g., EP0495257). Commercially available cellulases that find use in thepresent include, but are not limited to CELLUZYME®, CAREZYME®(Novozymes), REVITALENZ™ 100 (Danisco US Inc) and KAC-500(B)™ (KaoCorporation). In some embodiments, cellulases are incorporated asportions or fragments of mature wild-type or variant cellulases, whereina portion of the N-terminus is deleted (See e.g., U.S. Pat. No.5,874,276). Additional suitable cellulases include those found inWO2005054475, WO2005056787, U.S. Pat. Nos. 7,449,318, and 7,833,773. Insome embodiments, the cleaning compositions of the present inventionfurther comprise cellulases at a level from about 0.00001 to about 10%of additional cellulase by weight of the composition and the balance ofcleaning adjunct materials by weight of composition. In some otherembodiments of the present invention, the cleaning compositions of thepresent invention also comprise cellulases at a level of about 0.0001 toabout 10%, about 0.001 to about 5%, about 0.001 to about 2%, about 0.005to about 0.5% cellulase by weight of the composition.

Any mannanase suitable for use in detergent compositions also finds usein the present invention. Suitable mannanases include, but are notlimited to those of bacterial or fungal origin. Chemically orgenetically modified mutants are included in some embodiments. Variousmannanases are known which find use in the present invention (See e.g.,U.S. Pat. Nos. 6,566,114; 6,602,842; and 6,440,991, all of which areincorporated herein by reference). Commercially available mannanasesthat find use in the present invention include, but are not limited toMANNASTAR®, PURABRITE™, and MANNAWAY®. In some embodiments, the cleaningcompositions of the present invention further comprise mannanases at alevel from about 0.00001 to about 10% of additional mannanase by weightof the composition and the balance of cleaning adjunct materials byweight of composition. In some embodiments of the present invention, thecleaning compositions of the present invention also comprise mannanasesat a level of about 0.0001 to about 10%, about 0.001 to about 5%, about0.001 to about 2%, about 0.005 to about 0.5% mannanase by weight of thecomposition.

In some embodiments, peroxidases are used in combination with hydrogenperoxide or a source thereof (e.g., a percarbonate, perborate orpersulfate) in the compositions of the present invention. In somealternative embodiments, oxidases are used in combination with oxygen.Both types of enzymes are used for “solution bleaching” (i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen the fabrics are washed together in a wash liquor), preferablytogether with an enhancing agent (See e.g., WO94/12621 and WO95/01426).Suitable peroxidases/oxidases include, but are not limited to those ofplant, bacterial or fungal origin. Chemically or genetically modifiedmutants are included in some embodiments. In some embodiments, thecleaning compositions of the present invention further compriseperoxidase and/or oxidase enzymes at a level from about 0.00001 to about10% of additional peroxidase and/or oxidase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In some other embodiments of the present invention, the cleaningcompositions of the present invention also comprise peroxidase and/oroxidase enzymes at a level of about 0.0001 to about 10%, about 0.001 toabout 5%, about 0.001 to about 2%, about 0.005 to about 0.5% peroxidaseand/or oxidase enzymes by weight of the composition.

In some embodiments, additional enzymes find use, including but notlimited to perhydrolases (See e.g., WO2005056782, WO2007106293,WO2008063400, WO2008106214, and WO2008106215). In addition, in someembodiments, mixtures of the above mentioned enzymes are encompassedherein, in particular one or more additional protease, amylase, lipase,mannanase, and/or at least one cellulase. Indeed, it is contemplatedthat various mixtures of these enzymes will find use in the presentinvention. It is also contemplated that the varying levels of the serineprotease polypeptide (s) and one or more additional enzymes may bothindependently range to about 10%, the balance of the cleaningcomposition being cleaning adjunct materials. The specific selection ofcleaning adjunct materials are readily made by considering the surface,item, or fabric to be cleaned, and the desired form of the compositionfor the cleaning conditions during use (e.g., through the wash detergentuse).

In some embodiments, an effective amount of one or more serine proteasepolypeptide (s) provided herein is included in compositions useful forcleaning a variety of surfaces in need of proteinaceous stain removal.Such cleaning compositions include cleaning compositions for suchapplications as cleaning hard surfaces, fabrics, and dishes. Indeed, insome embodiments, the present invention provides fabric cleaningcompositions, while in other embodiments the present invention providesnon-fabric cleaning compositions. Notably, the present invention alsoprovides cleaning compositions suitable for personal care, includingoral care (including dentrifices, toothpastes, mouthwashes, etc., aswell as denture cleaning compositions), skin, and hair cleaningcompositions. It is intended that the present invention encompassdetergent compositions in any form (i.e., liquid, granular, bar,semi-solid, gels, emulsions, tablets, capsules, etc.).

By way of example, several cleaning compositions wherein the serineprotease polypeptides of the present invention find use are described ingreater detail below. In some embodiments in which the cleaningcompositions of the present invention are formulated as compositionssuitable for use in laundry machine washing method(s), the compositionsof the present invention preferably contain at least one surfactant andat least one builder compound, as well as one or more cleaning adjunctmaterials preferably selected from organic polymeric compounds,bleaching agents, additional enzymes, suds suppressors, dispersants,lime-soap dispersants, soil suspension and anti-redeposition agents andcorrosion inhibitors. In some embodiments, laundry compositions alsocontain softening agents (i.e., as additional cleaning adjunctmaterials). The compositions of the present invention also find use indetergent additive products in solid or liquid form. Such additiveproducts are intended to supplement and/or boost the performance ofconventional detergent compositions and can be added at any stage of thecleaning process. In some embodiments, the density of the laundrydetergent compositions herein ranges from about 400 to about 1200g/liter, while in other embodiments it ranges from about 500 to about950 g/liter of composition measured at 20° C.

In embodiments formulated as compositions for use in manual dishwashingmethods, the compositions of the invention preferably contain at leastone surfactant and preferably at least one additional cleaning adjunctmaterial selected from organic polymeric compounds, suds enhancingagents, group II metal ions, solvents, hydrotropes and additionalenzymes.

In some embodiments, various cleaning compositions such as thoseprovided in U.S. Pat. No. 6,605,458, find use with the serine proteasepolypeptides of the present invention. Thus, in some embodiments, thecompositions comprising at least one serine protease polypeptide of thepresent invention is a compact granular fabric cleaning composition,while in other embodiments, the composition is a granular fabriccleaning composition useful in the laundering of colored fabrics, infurther embodiments, the composition is a granular fabric cleaningcomposition which provides softening through the wash capacity, inadditional embodiments, the composition is a heavy duty liquid fabriccleaning composition. In some embodiments, the compositions comprisingat least one serine protease polypeptide of the present invention arefabric cleaning compositions such as those described in U.S. Pat. Nos.6,610,642 and 6,376,450. In addition, the serine protease polypeptidesof the present invention find use in granular laundry detergentcompositions of particular utility under European or Japanese washingconditions (See e.g., U.S. Pat. No. 6,610,642).

In some alternative embodiments, the present invention provides hardsurface cleaning compositions comprising at least one serine proteasepolypeptide provided herein. Thus, in some embodiments, the compositionscomprising at least one serine protease polypeptide of the presentinvention is a hard surface cleaning composition such as those describedin U.S. Pat. Nos. 6,610,642; 6,376,450; and 6,376,450.

In yet further embodiments, the present invention provides dishwashingcompositions comprising at least one serine protease polypeptideprovided herein. Thus, in some embodiments, the compositions comprisingat least one serine protease polypeptide of the present invention is ahard surface cleaning composition such as those in U.S. Pat. Nos.6,610,642 and 6,376,450. In some still further embodiments, the presentinvention provides dishwashing compositions comprising at least oneserine protease polypeptide provided herein. In some furtherembodiments, the compositions comprising at least one serine proteasepolypeptide of the present invention comprise oral care compositionssuch as those in U.S. Pat. Nos. 6,376,450 and 6,376,450. Theformulations and descriptions of the compounds and cleaning adjunctmaterials contained in the aforementioned U.S. Pat. Nos. 6,376,450;6,605,458; 6,605,458; and 6,610,642 find use with the serine proteasepolypeptides provided herein.

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in U.S. Pat. Nos.5,879,584; 5,691,297; 5,574,005; 5,569,645; 5,565,422; 5,516,448;5,489,392; and 5,486,303, all of which are incorporated herein byreference. When a low pH cleaning composition is desired, the pH of suchcomposition is adjusted via the addition of a material such asmonoethanolamine or an acidic material such as HCl.

In some embodiments, the cleaning compositions according to the presentinvention comprise an acidifying particle or an amino carboxylicbuilder. Examples of an amino carboxylic builder include aminocarboxylicacids, salts and derivatives thereof. In some embodiment, the aminocarboxylic builder is an aminopolycarboxylic builder, such asglycine-N,N-diacetic acid or derivative of general formulaMOOC—CHR—N(CH2COOM)₂ where R is C₁₋₁₂alkyl and M is alkali metal. Insome embodiments, the amino carboxylic builder can be methylglycinediacetic acid (MGDA), GLDA (glutamic-N,N-diacetic acid), iminodisuccinicacid (IDS), carboxymethyl inulin and salts and derivatives thereof,aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid(ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid(IDA), N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl)asparticacid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), IDS (iminodiacetic acid) and salts and derivativesthereof such as N-methyliminodiacetic acid (MIDA),alpha-alanine-N,N-diacetic acid (alpha-ALDA), serine-N,N-diacetic acid(SEDA), isoserine-N,Ndiacetic acid (ISDA), phenylalanine-N,N-diaceticacid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA) and alkali metal salts andderivative thereof. In some embodiments, the acidifying particle has aweight geometric mean particle size of from about 400μ to about 1200μand a bulk density of at least 550 g/L. In some embodiments, theacidifying particle comprises at least about 5% of the builder.

In some embodiments, the acidifying particle can comprise any acid,including organic acids and mineral acids. Organic acids can have one ortwo carboxyls and in some instances up to 15 carbons, especially up to10 carbons, such as formic, acetic, propionic, capric, oxalic, succinic,adipic, maleic, fumaric, sebacic, malic, lactic, glycolic, tartaric andglyoxylic acids. In some embodiments, the acid is citric acid. Mineralacids include hydrochloric and sulphuric acid. In some instances, theacidifying particle of the invention is a highly active particlecomprising a high level of amino carboxylic builder. Sulphuric acid hasbeen found to further contribute to the stability of the final particle.

In some embodiments, the cleaning compositions according to the presentinvention comprise at least one surfactant and/or a surfactant systemwherein the surfactant is selected from nonionic surfactants, anionicsurfactants, cationic surfactants, ampholytic surfactants, zwitterionicsurfactants, semi-polar nonionic surfactants and mixtures thereof. Insome embodiments, the surfactant is present at a level of from about 0.1to about 60%, while in alternative embodiments the level is from about 1to about 50%, while in still further embodiments the level is from about5 to about 40%, by weight of the cleaning composition.

In some embodiments, the cleaning compositions of the present inventioncomprise one or more detergent builders or builder systems. In someembodiments incorporating at least one builder, the cleaningcompositions comprise at least about 1%, from about 3% to about 60% oreven from about 5% to about 40% builder by weight of the cleaningcomposition. Builders include, but are not limited to, the alkali metal,ammonium and alkanolammonium salts of polyphosphates, alkali metalsilicates, alkaline earth and alkali metal carbonates, aluminosilicates,polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers ofmaleic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-trihydroxybenzene-2, 4, 6-trisulphonic acid, and carboxymethyloxysuccinic acid,the various alkali metal, ammonium and substituted ammonium salts ofpolyacetic acids such as ethylenediamine tetraacetic acid andnitrilotriacetic acid, as well as polycarboxylates such as melliticacid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, andsoluble salts thereof. Indeed, it is contemplated that any suitablebuilder will find use in various embodiments of the present invention.

In some embodiments, the builders form water-soluble hardness ioncomplexes (e.g., sequestering builders), such as citrates andpolyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphospatehexahydrate, potassium tripolyphosphate, and mixed sodium and potassiumtripolyphosphate, etc.). It is contemplated that any suitable builderwill find use in the present invention, including those known in the art(See e.g., EP2100949).

In some embodiments, builders for use herein include phosphate buildersand non-phosphate builders. In some embodiments, the builder is aphosphate builder. In some embodiments, the builder is a non-phosphatebuilder. If present, builders are used in a level of from 0.1 to 80%, orfrom 5 to 60%, or from 10 to 50% by weight of the composition. In someembodiments the product comprises a mixture of phosphate andnon-phosphate builders. Suitable phosphate builders includemono-phosphates, di-phosphates, tri-polyphosphates oroligomeric-polyphosphates, including the alkali metal salts of thesecompounds, including the sodium salts. In some embodiments, a buildercan be sodium tripolyphosphate (STPP). Additionally, the composition cancomprise carbonate and/or citrate, preferably citrate that helps toachieve a neutral pH composition of the invention. Other suitablenon-phosphate builders include homopolymers and copolymers ofpolycarboxylic acids and their partially or completely neutralizedsalts, monomeric polycarboxylic acids and hydroxycarboxylic acids andtheir salts. In some embodiments, salts of the above mentioned compoundsinclude the ammonium and/or alkali metal salts, i.e. the lithium,sodium, and potassium salts, including sodium salts. Suitablepolycarboxylic acids include acyclic, alicyclic, hetero-cyclic andaromatic carboxylic acids, wherein in some embodiments, they can containat least two carboxyl groups which are in each case separated from oneanother by, in some instances, no more than two carbon atoms.

In some embodiments, the cleaning compositions of the present inventioncontain at least one chelating agent. Suitable chelating agents include,but are not limited to copper, iron and/or manganese chelating agentsand mixtures thereof. In embodiments in which at least one chelatingagent is used, the cleaning compositions of the present inventioncomprise from about 0.1 to about 15% or even from about 3.0 to about 10%chelating agent by weight of the subject cleaning composition.

In some still further embodiments, the cleaning compositions providedherein contain at least one deposition aid. Suitable deposition aidsinclude, but are not limited to, polyethylene glycol, polypropyleneglycol, polycarboxylate, soil release polymers such as polytelephthalicacid, clays such as kaolinite, montmorillonite, atapulgite, illite,bentonite, halloysite, and mixtures thereof.

As indicated herein, in some embodiments, anti-redeposition agents finduse in some embodiments of the present invention. In some embodiments,non-ionic surfactants find use. For example, in automatic dishwashingembodiments, non-ionic surfactants find use for surface modificationpurposes, in particular for sheeting, to avoid filming and spotting andto improve shine. These non-ionic surfactants also find use inpreventing the re-deposition of soils. In some embodiments, theanti-redeposition agent is a non-ionic surfactant as known in the art(See e.g., EP2100949). In some embodiments, the non-ionic surfactant canbe ethoxylated nonionic surfactants, epoxy-capped poly(oxyalkylated)alcohols and amine oxides surfactants.

In some embodiments, the cleaning compositions of the present inventioninclude one or more dye transfer inhibiting agents. Suitable polymericdye transfer inhibiting agents include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof. In embodiments in which atleast one dye transfer inhibiting agent is used, the cleaningcompositions of the present invention comprise from about 0.0001 toabout 10%, from about 0.01 to about 5%, or even from about 0.1 to about3% by weight of the cleaning composition.

In some embodiments, silicates are included within the compositions ofthe present invention. In some such embodiments, sodium silicates (e.g.,sodium disilicate, sodium metasilicate, and crystalline phyllosilicates)find use. In some embodiments, silicates are present at a level of fromabout 1 to about 20%. In some embodiments, silicates are present at alevel of from about 5 to about 15% by weight of the composition.

In some still additional embodiments, the cleaning compositions of thepresent invention also contain dispersants. Suitable water-solubleorganic materials include, but are not limited to the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

In some further embodiments, the enzymes used in the cleaningcompositions are stabilized by any suitable technique. In someembodiments, the enzymes employed herein are stabilized by the presenceof water-soluble sources of calcium and/or magnesium ions in thefinished compositions that provide such ions to the enzymes. In someembodiments, the enzyme stabilizers include oligosaccharides,polysaccharides, and inorganic divalent metal salts, including alkalineearth metals, such as calcium salts, such as calcium formate. It iscontemplated that various techniques for enzyme stabilization will finduse in the present invention. For example, in some embodiments, theenzymes employed herein are stabilized by the presence of water-solublesources of zinc (II), calcium (II) and/or magnesium (II) ions in thefinished compositions that provide such ions to the enzymes, as well asother metal ions (e.g., barium (II), scandium (II), iron (II), manganese(II), aluminum (III), Tin (II), cobalt (II), copper (II), nickel (II),and oxovanadium (IV). Chlorides and sulfates also find use in someembodiments of the present invention. Examples of suitableoligosaccharides and polysaccharides (e.g., dextrins) are known in theart (See e.g., WO07/145964). In some embodiments, reversible proteaseinhibitors also find use, such as boron-containing compounds (e.g.,borate, 4-formyl phenyl boronic acid) and/or a tripeptide aldehyde finduse to further improve stability, as desired.

In some embodiments, bleach, bleach activators and/or bleach catalystsare present in the compositions of the present invention. In someembodiments, the cleaning compositions of the present invention compriseinorganic and/or organic bleaching compound(s). Inorganic bleachesinclude, but are not limited to perhydrate salts (e.g., perborate,percarbonate, perphosphate, persulfate, and persilicate salts). In someembodiments, inorganic perhydrate salts are alkali metal salts. In someembodiments, inorganic perhydrate salts are included as the crystallinesolid, without additional protection, although in some otherembodiments, the salt is coated. Any suitable salt known in the artfinds use in the present invention (See e.g., EP2100949).

In some embodiments, bleach activators are used in the compositions ofthe present invention. Bleach activators are typically organic peracidprecursors that enhance the bleaching action in the course of cleaningat temperatures of 60° C. and below. Bleach activators suitable for useherein include compounds which, under perhydrolysis conditions, givealiphatic peroxoycarboxylic acids having preferably from about 1 toabout 10 carbon atoms, in particular from about 2 to about 4 carbonatoms, and/or optionally substituted perbenzoic acid. Additional bleachactivators are known in the art and find use in the present invention(See e.g., EP2100949).

In addition, in some embodiments and as further described herein, thecleaning compositions of the present invention further comprise at leastone bleach catalyst. In some embodiments, the manganesetriazacyclononane and related complexes find use, as well as cobalt,copper, manganese, and iron complexes. Additional bleach catalysts finduse in the present invention (See e.g., U.S. Pat. Nos. 4,246,612;5,227,084; and 4,810,410; WO99/06521; and EP2100 949).

In some embodiments, the cleaning compositions of the present inventioncontain one or more catalytic metal complexes. In some embodiments, ametal-containing bleach catalyst finds use. In some embodiments, themetal bleach catalyst comprises a catalyst system comprising atransition metal cation of defined bleach catalytic activity, (e.g.,copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganesecations), an auxiliary metal cation having little or no bleach catalyticactivity (e.g., zinc or aluminum cations), and a sequestrate havingdefined stability constants for the catalytic and auxiliary metalcations, particularly ethylenediaminetetraacetic acid,ethylenediaminetetra (methylenephosphonic acid) and water-soluble saltsthereof are used (See e.g., U.S. Pat. No. 4,430,243). In someembodiments, the cleaning compositions of the present invention arecatalyzed by means of a manganese compound. Such compounds and levels ofuse are well known in the art (See e.g., U.S. Pat. No. 5,576,282). Inadditional embodiments, cobalt bleach catalysts find use in the cleaningcompositions of the present invention. Various cobalt bleach catalystsare known in the art (See e.g., U.S. Pat. Nos. 5,597,936 and 5,595,967)and are readily prepared by known procedures.

In some additional embodiments, the cleaning compositions of the presentinvention include a transition metal complex of a macropolycyclic rigidligand (MRL). As a practical matter, and not by way of limitation, insome embodiments, the compositions and cleaning processes provided bythe present invention are adjusted to provide on the order of at leastone part per hundred million of the active MRL species in the aqueouswashing medium, and in some embodiments, provide from about 0.005 ppm toabout 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, andmost preferably from about 0.1 ppm to about 5 ppm, of the MRL in thewash liquor.

In some embodiments, transition-metals in the instant transition-metalbleach catalyst include, but are not limited to manganese, iron andchromium. MRLs also include, but are not limited to special ultra-rigidligands that are cross-bridged (e.g.,5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitabletransition metal MRLs are readily prepared by known procedures (Seee.g., WO2000/32601 and U.S. Pat. No. 6,225,464).

In some embodiments, the cleaning compositions of the present inventioncomprise metal care agents. Metal care agents find use in preventingand/or reducing the tarnishing, corrosion, and/or oxidation of metals,including aluminum, stainless steel, and non-ferrous metals (e.g.,silver and copper). Suitable metal care agents include those describedin EP2100 949, WO9426860 and WO94/26859). In some embodiments, the metalcare agent is a zinc salt. In some further embodiments, the cleaningcompositions of the present invention comprise from about 0.1% to about5% by weight of one or more metal care agent.

In some embodiments, the cleaning composition is a high density liquid(HDL) composition having a variant serine protease polypeptide protease.The HDL liquid laundry detergent can comprise a detersive surfactant(10%-40%) comprising anionic detersive surfactant (selected from a groupof linear or branched or random chain, substituted or unsubstitutedalkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkylphosphates, alkyl phosphonates, alkyl carboxylates, and/or mixturesthereof); and optionally non-ionic surfactant (selected from a group oflinear or branched or random chain, substituted or unsubstituted alkylalkoxylated alcohol, for example a C₈-C₁₈ alkyl ethoxylated alcoholand/or C₆-C₁₂ alkyl phenol alkoxylates), optionally wherein the weightratio of anionic detersive surfactant (with a hydrophilic index (HIc) offrom 6.0 to 9) to non-ionic detersive surfactant is greater than 1:1.

The composition can comprise optionally, a surfactancy boosting polymerconsisting of amphiphilic alkoxylated grease cleaning polymers (selectedfrom a group of alkoxylated polymers having branched hydrophilic andhydrophobic properties, such as alkoxylated polyalkylenimines in therange of 0.05 wt %-10 wt %) and/or random graft polymers (typicallycomprising of hydrophilic backbone comprising monomers selected from thegroup consisting of: unsaturated C₁-C₆ carboxylic acids, ethers,alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleicanhydride, saturated polyalcohols such as glycerol, and mixturesthereof; and hydrophobic side chain(s) selected from the groupconsisting of: C₄-C₂₅ alkyl group, polypropylene, polybutylene, vinylester of a saturated C₂-C₆ mono-carboxylic acid, C₁-C₆ alkyl ester ofacrylic or methacrylic acid, and mixtures thereof.

The composition can comprise additional polymers such as soil releasepolymers (include anionically end-capped polyesters, for example SRP1,polymers comprising at least one monomer unit selected from saccharide,dicarboxylic acid, polyol and combinations thereof, in random or blockconfiguration, ethylene terephthalate-based polymers and co-polymersthereof in random or block configuration, for example Repel-o-tex SF,SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300and SRN325, Marloquest SL), anti-redeposition polymers (0.1 wt % to 10wt %, include carboxylate polymers, such as polymers comprising at leastone monomer selected from acrylic acid, maleic acid (or maleicanhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid,citraconic acid, methylenemalonic acid, and any mixture thereof,vinylpyrrolidone homopolymer, and/or polyethylene glycol, molecularweight in the range of from 500 to 100,000 Da); cellulosic polymer(including those selected from alkyl cellulose, alkyl alkoxyalkylcellulose, carboxyalkyl cellulose, alkyl carboxyalkyl cellulose examplesof which include carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixturesthereof) and polymeric carboxylate (such as maleate/acrylate randomcopolymer or polyacrylate homopolymer).

The composition can further comprise saturated or unsaturated fattyacid, preferably saturated or unsaturated C₁₂-C₂₄ fatty acid (0 wt % to10 wt %); deposition aids (examples for which include polysaccharides,preferably cellulosic polymers, poly diallyl dimethyl ammonium halides(DADMAC), and co-polymers of DAD MAC with vinyl pyrrolidone,acrylamides, imidazoles, imidazolinium halides, and mixtures thereof, inrandom or block configuration, cationic guar gum, cationic cellulosesuch as cationic hydroxyethyl cellulose, cationic starch, cationicpolyacrylamides, and mixtures thereof.

The composition can further comprise dye transfer inhibiting agentsexamples of which include manganese phthalocyanine, peroxidases,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles and/or mixtures thereof; chelating agents examplesof which include ethylene-diamine-tetraacetic acid (EDTA); diethylenetriamine penta methylene phosphonic acid (DTPMP); hydroxy-ethanediphosphonic acid (HEDP); ethylenediamine N,N′-disuccinic acid (EDDS);methyl glycine diacetic acid (MGDA); diethylene triamine penta aceticacid (DTPA); propylene diamine tetracetic acid (PDT A);2-hydroxypyridine-N-oxide (HPNO); or methyl glycine diacetic acid(MGDA); glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl glutamicacid tetrasodium salt (GLDA); nitrilotriacetic acid (NTA);4,5-dihydroxy-m-benzenedisulfonic acid; citric acid and any saltsthereof; N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP) and derivatives thereof.

The composition may comprise an enzyme stabilizer (examples of whichinclude polyols such as propylene glycol or glycerol, sugar or sugaralcohol, lactic acid, reversible protease inhibitor, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid).

The composition can further comprise silicone or fatty-acid based sudssuppressors; heuing dyes, calcium and magnesium cations, visualsignaling ingredients, anti-foam (0.001 wt % to about 4.0 wt %), and/orstructurant/thickener (0.01 wt % to 5 wt %, selected from the groupconsisting of diglycerides and triglycerides, ethylene glycoldistearate, microcrystalline cellulose, cellulose based materials,microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixturesthereof).

Suitable detersive surfactants also include cationic detersivesurfactants (selected from a group of alkyl pyridinium compounds, alkylquaternary ammonium compounds, alkyl quaternary phosphonium compounds,alkyl ternary sulphonium compounds, and/or mixtures thereof);zwitterionic and/or amphoteric detersive surfactants (selected from agroup of alkanolamine sulpho-betaines); ampholytic surfactants;semi-polar non-ionic surfactants and mixtures thereof.

The composition can be any liquid form, for example a liquid or gelform, or any combination thereof. The composition may be in any unitdose form, for example a pouch.

In some embodiments, the cleaning composition is a high density powder(HDD) composition having a variant serine protease polypeptide protease.The HDD powder laundry detergent can comprise a detersive surfactantincluding anionic detersive surfactants (selected from a group of linearor branched or random chain, substituted or unsubstituted alkylsulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkylphosphates, alkyl phosphonates, alkyl carboxylates and/or mixturesthereof), non-ionic detersive surfactant (selected from a group oflinear or branched or random chain, substituted or unsubstituted C₈-C₁₈alkyl ethoxylates, and/or C₆-C₁₂ alkyl phenol alkoxylates), cationicdetersive surfactants (selected from a group of alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof), zwitterionic and/or amphoteric detersive surfactants (selectedfrom a group of alkanolamine sulpho-betaines); ampholytic surfactants;semi-polar non-ionic surfactants and mixtures thereof; builders(phosphate free builders [for example zeolite builders examples of whichinclude zeolite A, zeolite X, zeolite P and zeolite MAP in the range of0 wt % to less than 10 wt %]; phosphate builders [examples of whichinclude sodium tri-polyphosphate in the range of 0 wt % to less than 10wt %]; citric acid, citrate salts and nitrilotriacetic acid or saltthereof in the range of less than 15 wt %); silicate salt (sodium orpotassium silicate or sodium meta-silicate in the range of 0 wt % toless than 10 wt %, or layered silicate (SKS-6)); carbonate salt (sodiumcarbonate and/or sodium bicarbonate in the range of 0 wt % to less than10 wt %); and bleaching agents (photobleaches, examples of which includesulfonated zinc phthalocyanines, sulfonated aluminum phthalocyanines,xanthenes dyes, and mixtures thereof; hydrophobic or hydrophilic bleachactivators (examples of which include dodecanoyl oxybenzene sulfonate,decanoyl oxybenzene sulfonate, decanoyl oxybenzoic acid or saltsthereof, 3,5,5-trimethyl hexanoyl oxybenzene sulfonate, tetraacetylethylene diamine-TAED, and nonanoyloxybenzene sulfonate-NOBS, nitrilequats, and mixtures thereof; hydrogen peroxide; sources of hydrogenperoxide (inorganic perhydrate salts examples of which include mono ortetra hydrate sodium salt of perborate, percarbonate, persulfate,perphosphate, or persilicate); preformed hydrophilic and/or hydrophobicperacids (selected from a group consisting of percarboxylic acids andsalts, percarbonic acids and salts, perimidic acids and salts,peroxymonosulfuric acids and salts) & mixtures thereof and/or bleachcatalyst (such as imine bleach boosters examples of which includeiminium cations and polyions; iminium zwitterions; modified amines;modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones andmixtures thereof; metal-containing bleach catalyst for example copper,iron, titanium, ruthenium, tungsten, molybdenum, or manganese cationsalong with an auxiliary metal cations such as zinc or aluminum and asequestrate such as ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof).

The composition can further comprise additional detergent ingredientsincluding perfume microcapsules, starch encapsulated perfume accord,hueing agents, additional polymers including fabric integrity andcationic polymers, dye lock ingredients, fabric-softening agents,brighteners (for example C.I. Fluorescent brighteners), flocculatingagents, chelating agents, alkoxylated polyamines, fabric depositionaids, and/or cyclodextrin.

In some embodiments, the cleaning composition is an automaticdishwashing (ADW) detergent composition having a serine protease of thepresent invention. The ADW detergent composition can comprise two ormore non-ionic surfactants selected from a group of ethoxylatednon-ionic surfactants, alcohol alkoxylated surfactants, epoxy-cappedpoly(oxyalkylated) alcohols, or amine oxide surfactants present inamounts from 0 to 10% by weight; builders in the range of 5-60%comprising either phosphate (mono-phosphates, di-phosphates,tri-polyphosphates or oligomeric-poylphosphates, preferred sodiumtripolyphosphate-STPP or phosphate-free builders [amino acid basedcompounds, examples of which include MGDA (methyl-glycine-diaceticacid), and salts and derivatives thereof, GLDA (glutamic-N,Ndiaceticacid) and salts and derivatives thereof, IDS (iminodisuccinic acid) andsalts and derivatives thereof, carboxy methyl inulin and salts andderivatives thereof and mixtures thereof, nitrilotriacetic acid (NTA),diethylene triamine penta acetic acid (DTPA), B-alaninediacetic acid(B-ADA) and their salts], homopolymers and copolymers of poly-carboxylicacids and their partially or completely neutralized salts, monomericpolycarboxylic acids and hydroxycarboxylic acids and their salts in therange of 0.5% to 50% by weight; sulfonated/carboxylated polymers(provide dimensional stability to the product) in the range of about0.1% to about 50% by weight; drying aids in the range of about 0.1% toabout 10% by weight (selected from polyesters, especially anionicpolyesters optionally together with further monomers with 3 to 6functionalities which are conducive to polycondensation, specificallyacid, alcohol or ester functionalities, polycarbonate-, polyurethane-and/or polyurea-polyorganosiloxane compounds or precursor compoundsthereof of the reactive cyclic carbonate and urea type); silicates inthe range from about 1% to about 20% by weight (sodium or potassiumsilicates for example sodium disilicate, sodium meta-silicate andcrystalline phyllosilicates); bleach-inorganic (for example perhydratesalts such as perborate, percarbonate, perphosphate, persulfate andpersilicate salts) and organic (for example organic peroxyacidsincluding diacyl and tetraacylperoxides, especially diperoxydodecanediocacid, diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid);bleach activators—organic peracid precursors in the range from about0.1% to about 10% by weight; bleach catalysts (selected from manganesetriazacyclononane and related complexes, Co, Cu, Mn and Febispyridylamine and related complexes, and pentamine acetate cobalt(III)and related complexes); metal care agents in the range from about 0.1%to 5% by weight (selected from benzatriazoles, metal salts andcomplexes, and/or silicates); enzymes in the range from about 0.01 to5.0 mg of active enzyme per gram of automatic dishwashing detergentcomposition (acyl transferases, alpha-amylases, beta-amylases,alpha-galactosidases, arabinosidases, aryl esterases,beta-galactosidases, carrageenases, catalases, cellobiohydrolases,cellulases, chondroitinases, cutinases, endo-beta-1, 4-glucanases,endo-beta-mannanases, esterases, exo-mannanases, galactanases,glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases,lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases,pectate lyases, pectin acetyl esterases, pectinases, pentosanases,peroxidases, phenoloxidases, phosphatases, phospholipases, phytases,polygalacturonases, proteases, pullulanases, reductases,rhamnogalacturonases, beta-glucanases, tannases, transglutaminases,xylan acetyl-esterases, xylanases, xyloglucanases, and xylosidases, andany mixture thereof); and enzyme stabilizer components (selected fromoligosaccharides, polysaccharides and inorganic divalent metal salts).

In some embodiments, the cleaning composition is borate-free. In someembodiments, the cleaning composition is phosphate-free.

Representative detergent formulations that beneficially include a serineprotease polypeptide of the present invention include the detergentformulations found in WO2013063460, pages 78-152, and in particular thetables of pages 94 to 152 are hereby incorporated by reference. Theserine proteases are normally incorporated into the detergentcomposition at a level of from 0.00001% to 10% of enzyme protein byweight of the composition. In some embodiments, the detergentcomposition comprises more than 0.0001%, 0.001%, 0.01%, or 0.1% of theserine protease by weight of the composition. In some embodiments, thedetergent composition comprises less than 1%, 0.1%, 0.01%, or 0.001% ofthe serine protease by weight of the composition.

Also provided are compositions and methods of treating fabrics (e.g., todesize a textile) using a serine protease polypeptide of the presentinvention. Fabric-treating methods are well known in the art (see, e.g.,U.S. Pat. No. 6,077,316). For example, the feel and appearance of afabric can be improved by a method comprising contacting the fabric witha serine protease in a solution. The fabric can be treated with thesolution under pressure.

A serine protease of the present invention can be applied during orafter the weaving of a textile, or during the desizing stage, or one ormore additional fabric processing steps. During the weaving of textiles,the threads are exposed to considerable mechanical strain. Prior toweaving on mechanical looms, warp yarns are often coated with sizingstarch or starch derivatives to increase their tensile strength and toprevent breaking. A serine protease of the present invention can beapplied during or after the weaving to remove these sizing starch orstarch derivatives. After weaving, the serine protease can be used toremove the size coating before further processing the fabric to ensure ahomogeneous and wash-proof result.

A serine protease of the present invention can be used alone or withother desizing chemical reagents and/or desizing enzymes to desizefabrics, including cotton-containing fabrics, as detergent additives,e.g., in aqueous compositions. An amylase also can be used incompositions and methods for producing a stonewashed look on indigo-dyeddenim fabric and garments. For the manufacture of clothes, the fabriccan be cut and sewn into clothes or garments, which are afterwardsfinished. In particular, for the manufacture of denim jeans, differentenzymatic finishing methods have been developed. The finishing of denimgarment normally is initiated with an enzymatic desizing step, duringwhich garments are subjected to the action of proteolytic enzymes toprovide softness to the fabric and make the cotton more accessible tothe subsequent enzymatic finishing steps. The serine protease can beused in methods of finishing denim garments (e.g., a “bio-stoningprocess”), enzymatic desizing and providing softness to fabrics, and/orfinishing process.

The serine protease polypeptides described herein find further use inthe enzyme aided removal of proteins from animals and their subsequentdegradation or disposal, such as feathers, skin, hair, hide, and thelike. In some instances, immersion of the animal carcass in a solutioncomprising a serine protease polypeptide of the present invention canact to protect the skin from damage in comparison to the traditionalimmersion in scalding water or the defeathering process. In oneembodiment, feathers can be sprayed with an isolated serine protasepolypeptide of the present invention under conditions suitable fordigesting or initiating degradation of the plumage. In some embodiments,a serine protease of the present invention can be used, as above, incombination with an oxidizing agent.

In some embodiments, removal of the oil or fat associated with rawfeathers is assisted by using a serine protease polypeptide of thepresent invention. In some embodiments, the serine protease polypeptidesare used in compositions for cleaning the feathers as well as tosanitize and partially dehydrate the fibers. In yet other embodiments,the disclosed serine protease polypeptides find use in recoveringprotein from plumage. In some other embodiments, the serine proteasepolypeptides are applied in a wash solution in combination with 95%ethanol or other polar organic solvent with or without a surfactant atabout 0.5% (v/v).

In a further aspect of the invention, the serine protease polypeptidesof the present invention can be used as a component of an animal feedcomposition, animal feed additive and/or pet food comprising a serineprotease and variants thereof. The present invention further relates toa method for preparing such an animal feed composition, animal feedadditive composition and/or pet food comprising mixing the serineprotease polypeptide with one or more animal feed ingredients and/oranimal feed additive ingredients and/or pet food ingredients.Furthermore, the present invention relates to the use of the serineprotease polypeptide in the preparation of an animal feed compositionand/or animal feed additive composition and/or pet food.

The term “animal” includes all non-ruminant and ruminant animals. In aparticular embodiment, the animal is a non-ruminant animal, such as ahorse and a mono-gastric animal. Examples of mono-gastric animalsinclude, but are not limited to, pigs and swine, such as piglets,growing pigs, sows; poultry such as turkeys, ducks, chicken, broilerchicks, layers; fish such as salmon, trout, tilapia, catfish and carps;and crustaceans such as shrimps and prawns. In a further embodiment theanimal is a ruminant animal including, but not limited to, cattle, youngcalves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo,deer, camels, alpacas, llamas, antelope, pronghorn and nilgai.

In the present context, it is intended that the term “pet food” isunderstood to mean a food for a household animal such as, but notlimited to, dogs, cats, gerbils, hamsters, chinchillas, fancy rats,guinea pigs; avian pets, such as canaries, parakeets, and parrots;reptile pets, such as turtles, lizards and snakes; and aquatic pets,such as tropical fish and frogs.

The terms “animal feed composition,” “feedstuff” and “fodder” are usedinterchangeably and can comprise one or more feed materials selectedfrom the group comprising a) cereals, such as small grains (e.g., wheat,barley, rye, oats and combinations thereof) and/or large grains such asmaize or sorghum; b) by products from cereals, such as corn gluten meal,Distillers Dried Grain Solubles (DDGS) (particularly corn basedDistillers Dried Grain Solubles (cDDGS), wheat bran, wheat middlings,wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citruspulp; c) protein obtained from sources such as soya, sunflower, peanut,lupin, peas, fava beans, cotton, canola, fish meal, dried plasmaprotein, meat and bone meal, potato protein, whey, copra, sesame; d)oils and fats obtained from vegetable and animal sources; e) mineralsand vitamins.

The protease polypeptides described herein find further use in theenzyme aided bleaching of paper pulps such as chemical pulps,semi-chemical pulps, kraft pulps, mechanical pulps or pulps prepared bythe sulfite method. In general terms, paper pulps are incubated with aprotease polypeptide of the present invention under conditions suitablefor bleaching the paper pulp.

In some embodiments, the pulps are chlorine free pulps bleached withoxygen, ozone, peroxide or peroxyacids. In some embodiments, theprotease polypeptides are used in enzyme aided bleaching of pulpsproduced by modified or continuous pulping methods that exhibit lowlignin contents. In some other embodiments, the protease polypeptidesare applied alone or preferably in combination with xylanase and/orendoglucanase and/or alpha-galactosidase and/or cellobiohydrolaseenzymes.

The protease polypeptides described herein find further use in theenzyme aided removal of proteins from animals and their subsequentdegradation or disposal, such as feathers, skin, hair, hide, and thelike. In some instances, immersion of the animal carcass in a solutioncomprising a protease polypeptide of the present invention can act toprotect the skin from damage in comparison to the traditional immersionin scalding water or the defeathering process. In one embodiment,feathers can be sprayed with an isolated protease polypeptide of thepresent invention under conditions suitable for digesting or initiatingdegradation of the plumage. In some embodiments, a protease of thepresent invention can be used, as above, in combination with anoxidizing agent.

In some embodiments, removal of the oil or fat associated with rawfeathers is assisted by using a protease polypeptide of the presentinvention. In some embodiments, the protease polypeptides are used incompositions for cleaning the feathers as well as to sanitize andpartially dehydrate the fibers. In some other embodiments, the proteasepolypeptides are applied in a wash solution in combination with 95%ethanol or other polar organic solvent with or without a surfactant atabout 0.5% (v/v). In yet other embodiments, the disclosed proteasepolypeptides find use in recovering protein from plumage. The disclosedprotease polypeptides may be used alone or in combination in suitablefeather processing and proteolytic methods, such as those disclosed inPCT/EP2013/065362, PCT/EP2013/065363, and PCT/EP2013/065364, which arehereby incorporated by reference. In some embodiments, the recoveredprotein can be subsequently used in animal or fish feed.

EXAMPLES

The following examples are provided to demonstrate and illustratecertain preferred embodiments and aspects of the present disclosure andshould not be construed as limiting. In the experimental disclosurewhich follows, the following abbreviations apply: ADW (automatic dishwashing); BMI (blood/milk/ink); BSA (bovine serum albumin); CAPS(N-cyclohexyl-3-aminopropanesulfonic acid); CHES(N-cyclohexyl-2-aminoethanesulfonic acid); DMC (dimethyl casein); HDD(heavy duty dry/powder); HDL (heavy duty liquid); HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); MTP (microtiterplate); ND (not done); OD (optical density); PCR (polymerase chainreaction); ppm (parts per million); QS (quantity sufficient); rpm(revolutions per minute); AAPF(succinyl-Ala-Ala-Pro-Phe-p-nitroanilide); TNBSA (2,4,6-trinitrobenzenesulfonic acid); v/v (volume to volume); and w/v (weight to volume).

Example 1 Discovery and Identification of Serine Protease BspAP02013

Bacillus sp. 18N1 (Culture Collection Dupont) was selected as apotential source for enzymes useful in industrial applications. Toidentify enzymes produced by Bacillus sp. 18N1 and the genes that encodethese enzymes, the entire genome of Bacillus sp. 18N1 was sequencedusing Illumina® sequencing by synthesis (SBS) technology. Genomesequencing and assembly of the sequence data was performed by BaseClear(Leiden, The Netherlands). Contigs were annotated by BioXpr (Namur,Belgium). One of genes identified this way in strain Bacillus sp. 18N1encodes a protein that showed homology to serine proteases of variousother bacteria. The nucleotide sequence of this gene, BspAP02013.n, isdepicted in SEQ ID NO:1: TTGAGACGATTATTTTTAGCTCTATTACTAGTAATTTTGATGGCAGTACCGGGACAAGCTGTATTAGCAGGAAATGCGAACGAAGAGGTTAAAGATTATTTAGTTCAATTTAATGGTGCAGCACAGAAAGGGTTAGTACAAGCATTTGGTATTGATAACGAAGATATTATCCATGAATATGACCTCCTTCCAGTCATGCATCTAAATTTAACAGACAATCAAGCTCGTGGCCTGAAGAATCATCCTCATGTTCAAATGGTTGAAGAAAATGCTGAGGTAACGAAACTAGCTCAAACAACGCCATGGGGTATCCCTCGTGTTCAAGGAACTGCTGCACAAAATGCAGGCTATACAGGAAATGGGGTAAAGGTAGCGATTCTTGATACAGGAATTGATCGCAATCATCCTGATCTTTCTGCTAATGTAAAAGGTGGCCATTCTGTTTTCACTGATTCAGCTAACTCTGACCCATTTTTTGATGGTGATGGACACGGTACTCATGTTGCTGGTACTGTGGCGGCTGTGAATAATGATATTGGGGTTATTGGTGTCGCAAGTGAAGCTTCTCTATATGCGGTAAAGGTATTGAACAATGCGGGTAGTGGTTCATATGCTGGTATTGCCGAGGGAATCGAATGGGCAATCAATAATGATATCGACATCATTAATATGAGTCTTGGTGGCTCACAAAGTTCTGCTATTTTAAAACAGTTTAGTGATCTAGCATATGCTGAGGGACTCCTTGTTGTCGCTGCAGCTGGTAATAGTGGAACACGCAGTGGTAGAAATGACACAGTCGGCTACCCTGCTAAATATGACTCAGTTATCGCAGTAGCTGCAACGGATCAAAATAACCAACGTGCAACATTCTCAAGCACAGGTCCAGCAGTTGAAATCTCAGCTCCTGGAGTAGGCATTCTTAGCACGACGCCAAATAACAATTATGTATCCTTTAATGGAACATCAATGGCTTCTCCACACGTCGCAGGAGTTGCAGCGCAAGTGTGGCAAGCAAAGCCTCACTTATCGAATATTGAGCTTCGTAATCTGTTAAATGACACAGCTATTGATCTAGGATCTTCTACGCAATATGGTAATGGATTAGTACAATCATTAGAAGCGATTCAACAA.

The preproenzyme encoded by the BspAP02013.n gene is depicted in SEQ IDNO:2. At the N-terminus, the protein has a signal peptide with a lengthof 23 amino acids as predicted by SignalP-NN (Emanuelsson et al., NatureProtocols (2007) 2: 953-971). This signal peptide sequence is underlinedand in bold in SEQ ID NO:2. The presence of a signal peptide indicatesthat this serine protease is a secreted enzyme. The enzyme has a prosequence which is predicted to be 73 amino acids (shown in italics). Thesequence of the predicted, fully processed mature chain (BspAP02013, 281amino acids) is depicted in SEQ ID NO:3.

SEQ ID NO:2 sets forth the amino acid sequence of the serine proteaseprecursor BspAP02013 (the signal peptide sequence is underlined and inbold, the pro sequence is shown in italics):

LRRLFLALLLVILMAVPGQAVLA GNANEEVKDYLVQFNGAAQKGLVQAFGIDNEDIIHEYDLLPVMHLNLTDNQARGLKNHPHVQMVEENAEVTKLAQTTPWGIPRVQGTAAQNAGYTGNGVKVAILDTGIDRNHPDLSANVKGGHSVFTDSANSDPFFDGDGHGTHVAGTVAAVNNDIGVIGVASEASLYAVKVLNNAGSGSYAGIAEGIEWAINNDIDIINMSLGGSQSSAILKQFSDLAYAEGLLVVAAAGNSGTRSGRNDTVGYPAKYDSVIAVAATDQNNQRATFSSTGPAVEISAPGVGILSTTPNNNYVSFNGTSMASPHVAGVAAQVWQAKPHLSNIELRNLLNDTAIDLGSSTQYGNGLVQSLEAIQQ.

SEQ ID NO:3 sets forth the predicted amino acid sequence of the matureprotease BspAP02013: AQTTPWGIPRVQGTAAQNAGYTGNGVKVAILDTGIDRNHPDLSANVKGGHSVFTDSANSDPFFDGDGHGTHVAGTVAAVNNDIGVIGVASEASLYAVKVLNNAGSGSYAGIAEGIEWAINNDIDIINMSLGGSQSSAILKQFSDLAYAEGLLVVAAAGNSGTRSGRNDTVGYPAKYDSVIAVAATDQNNQRATFSSTGPAVEISAPGVGILSTTPNNNYVSFNGTSMASPHVAGVAAQVWQAKPHLSNIELRNLLNDTAIDLGSSTQYGNGLVQSLEAIQQ.

Example 2 Discovery and Identification of Serine Protease BspM02866

Bacillus sp. WDG290 (Culture Collection Dupont) was selected as apotential source for enzymes useful in industrial applications. Toidentify enzymes produced by Bacillus sp. WDG290 and the genes thatencode these enzymes, the entire genome of Bacillus sp. WDG290 wassequenced using Illumina® sequencing by synthesis (SBS) technology.Genome sequencing and assembly of the sequence data was performed byBaseClear (Leiden, The Netherlands). Contigs were annotated by BioXpr(Namur, Belgium). One of genes identified this way in strain Bacillussp. WDG290 encodes a protein that showed homology to serine proteases ofvarious other bacteria.

SEQ ID NO:4 sets forth the nucleotide sequence of the BspM02866.n gene:ATGAA GAAATTATTCGTAGTCTGGATGACGCTCATCTTAATGGCTGTGCCGTTTCAGGCAGGGGCCTCAAACGGATCCGGAGACACTCTCGAAGAATACTTAGTACAGTTTAACGGGCCTTCAGCGCATGGGCTGATGCAGGCATTCGGCATTGATGAAGCACAGGTGAAAACCGAATTCGATCACCTGCCGGTAGTAAATGTTGCCCTTTCTGAAGCTCAGGCAAGAGGCCTGGCGAACCACCCTCACGTAGAAGCGGTGGAGGAGAACGCTGAAGTGCATGCGCTTGGTCAGACGGTACCATGGGGCATTCCCCACGTTCAGGGAACGGCTGCCCAGGATGCAGGATTTACAGGAGCCGGTCTTAAGGTGGCAATTCTTGATACAGGAATTGAAGCATCCCACGAAGATCTGTCTGCGAACGTAAAAGGCGGGCACTCTGTTTTTACCGATTCTGCCAACAGTGATCCGTTCTACGATCCGAACGGACACGGCACACACGTTGCCGGAACGGTTGCAGCCGTCGATAACGATCTTGGTGTCATCGGCGTCGCTCCCGAAGCGGACCTTTACGCGGTTAAAGTACTCAGCAACGCCGGAAGCGGAAGCATCGCCGGCATTGCAGAGGGAATCGAATGGTCGATCGATAACGGAATGGATATCATTAATATGAGTCTGGGCGCTTCGCAGGGATCTTCCATCCTTGAGCAGTTCTCAAACCTTGCCTATGATGAAGGACTCCTTGTGGTGGCTGCTGCCGGTAACAGCGGAAACCGCGGCGGGAATAACAATACGGTCGGCTACCCGGCTGCCTATGACTCTGTTATTGCCGTAGCTGCGGTGGACCAGAACAACAATCGCGCCACGTTCTCCAGCACAGGCCCGGCTGTTGAAATCTCAGCACCCGGCGTCAACGTCCTCAGCACAACGCCTGGCAACAATTACGCTTCCTACAACGGAACGTCCATGGCGTCTCCTCACGTAGCAGGCGTAGCCGCCCAGGTATGGCAGGCGAATCCGGGGCTTTCCAACACAGAGCTCCGCCAGCTTCTCAATGATACAGCCGTCAACCTCGGCCCGGCCCACCAGTATGGTCACGGCCTAGTCCAGTCACTTGATGCGATTAACCAG.

The preproenzyme encoded by the BspM02866.n gene is depicted in SEQ IDNO:5. At the N-terminus, the protein has a signal peptide with a lengthof 22 amino acids as predicted by SignalP-NN (Emanuelsson et al., NatureProtocols (2007) 2:953-971). This signal peptide sequence is underlinedand in bold in SEQ ID NO:5. The presence of a signal peptide indicatesthat this serine protease is a secreted enzyme. The enzyme has a prosequence which is predicted to be 74 amino acids (shown in italics). Thesequence of the predicted, fully processed mature chain (BspM02866, 281amino acids) is depicted in SEQ ID NO:6.

SEQ ID NO:5 sets forth the amino acid sequence of the serine proteaseprecursor BspM02866 (the signal peptide sequence is underlined and inbold, the pro sequence is shown in italics):

MKKLFVVWMTLILMAVPFQAGA SNGSGDTLEEYLVQFNGPSAHGLMQAFGIDEAQVKTEFDHLPVVNVALSEAQARGLANHPHVEAVEENAEVHALGQTVPWGIPHVQGTAAQDAGFTGAGLKVAILDTGIEASHEDLSANVKGGHSVFTDSANSDPFYDPNGHGTHVAGTVAAVDNDLGVIGVAPEADLYAVKVLSNAGSGSIAGIAEGIEWSIDNGMDIINMSLGASQGSSILEQFSNLAYDEGLLVVAAAGNSGNRGGNNNTVGYPAAYDSVIAVAAVDQNNNRATFSSTGPAVEISAPGVNVLSTTPGNNYASYNGTSMASPHVAGVAAQVWQANPGLSNTELRQLLNDTAVNLGPAHQYGHGLVQSLDAINQ.

SEQ ID NO:6 sets forth the predicted amino acid sequence of the matureprotease BspM02866: GQTVPWGIPHVQGTAAQDAGFTGAGLKVAILDTGIEASHEDLSANVKGGHSVFTDSANSDPFYDPNGHGTHVAGTVAAVDNDLGVIGVAPEADLYAVKVLSNAGSGSIAGIAEGIEWSIDNGMDIINMSLGASQGSSILEQFSNLAYDEGLLVVAAAGNSGNRGGNNNTVGYPAAYDSVIAVAAVDQNNNRATFSSTGPAVEISAPGVNVLSTTPGNNYASYNGTSMASPHVAGVAAQVWQANPGLSNTELRQLLNDTAVNLGPAHQYGHGLVQSLDAINQ.

Example 3 Discovery and Identification of Serine Protease BspZ00056

Bacillus sp. WDG291 (Culture Collection Dupont) was selected as apotential source for enzymes useful in industrial applications. Toidentify enzymes produced by Bacillus sp. WDG291 and the genes thatencode these enzymes, the entire genome of Bacillus sp. WDG291 wassequenced using Illumina® sequencing by synthesis (SBS) technology.Genome sequencing and assembly of the sequence data was performed byBaseClear (Leiden, The Netherlands). Contigs were annotated by BioXpr(Namur, Belgium). One of genes identified this way in strain Bacillussp. WDG291 encodes a protein that showed homology to serine proteases ofvarious other bacteria.

SEQ ID NO:7 sets forth the nucleotide sequence of the BspZ00056.n gene:TTGAAA AAATTATTCACAGTTTGGTTAGCACTCGTTTTACTAGCGATTCCCGTATCTGTCGGGGCAGATGCTGGTGGTAACGATCAAAGTCAGGATTATTTGGTCCAATTTAACGGACCTGCAAGTAAAGGGTTAATTAAAGCGTTCGGTGTCGATGAAGGGGACATTCTTCATACATACGACCACCTTCCAGTCGTCCACGTGAACTTGACTGAAAACCAGGCACGGGGCCTTGCCAATCACCCACACATCACAACAGTTGAAGAAAACGCTGAAGTAAAAGCGCTCGGTCAAACGGTCCCATGGGGCATTCCACACGTGCAAGGAACTGCGGCTCAAGATGCTGGGTATACTGGTGCCGGTCTTAAAGTAGCGATTCTTGATACGGGGATCGACCGTAACCACGAAGACTTGTTTGCTAACGTAAAAGGCGGTCATTCCGTATTTACGGATTCCGCAAACAGCGATCCATTTTATGATGCTGACGGTCACGGTACACACGTTGCAGGTACAGTCGCAGCTGTTGATAACGACCTTGGCGTTGTAGGCGTGGCTTCCCAAGCTGAGCTGTATGCGGTAAAAGTTCTGAACAACTCCGGAAGCGGATCTTATGCAGGTATCGCTGAAGGAATTGAATGGTCGATCAACAACGGAATGGACATCATCAACATGAGCCTTGGTGGTTCCCAAAGCTCGTCCATCCTGAAACAGTTCTCTGACTTAGCTTACGAAGAAGGACTTCTTGTCGTAGCCGCAGCGGGTAACAGCGGAAACCGCGGTGGAAACAACGACACTGTCGGCTACCCGGCGAAATATGACTCTGTAATCGCGGTCGCTGCCGTCGATCAAAACAACAACCGTGCTACATTCTCTAGTACCGGTCCTGCTGTTGAAATTTCAGCTCCTGGTGTGAGCATTCTCAGCACAACGCCAGGCAACAACTACGCTGCGTTCAACGGAACTTCCATGGCTTCTCCTCACGTAGCCGGCGTGGCAGCTCAAGTTTGGCAGGCAAAACCTGAACTATCAAACGTAGAGCTTCGTAATCTATTAAACGAAACTGCAGTGAACCTGGGCGGATCCAACCAATTCGGTCACGGTCTAGTTCAGTCGCTGGATGCGATTCAGCAC.

The preproenzyme encoded by the BspZ00056.n gene is depicted in SEQ IDNO:8. At the N-terminus, the protein has a signal peptide with a lengthof 24 amino acids as predicted by SignalP-NN (Emanuelsson et al., NatureProtocols (2007) 2:953-971). This signal peptide sequence is underlinedand in bold in SEQ ID NO:8. The presence of a signal peptide indicatesthat this serine protease is a secreted enzyme. The enzyme has a prosequence which is predicted to be 72 amino acids (shown in italics). Thesequence of the predicted, fully processed mature chain (BspZ00056, 281amino acids) is depicted in SEQ ID NO:9.

SEQ ID NO:8 sets forth the amino acid sequence of the serine proteaseprecursor BspZ00056 (the signal peptide sequence is underlined and inbold, the pro sequence is shown in italics):

LKKLFTV W LALVLLAIPVSVGADA GGNDQSQDYLVQFNGPASKGLIKAFGVDEGDILHTYDHLPVVHVNLTENQARGLANHPHITTVEENAEVKALGQTVPWGIPHVQGTAAQDAGYTGAGLKVAILDTGIDRNHEDLFANVKGGHSVFTDSANSDPFYDADGHGTHVAGTVAAVDNDLGVVGVASQAELYAVKVLNNSGSGSYAGIAEGIEWSINNGMDIINMSLGGSQSSSILKQFSDLAYEEGLLVVAAAGNSGNRGGNNDTVGYPAKYDSVIAVAAVDQNNNRATFSSTGPAVEISAPGVSILSTTPGNNYAAFNGTSMASPHVAGVAAQVWQAKPELSNVELRNLLNETAVNLGGSNQFGHGLVQSLDAIQH.

SEQ ID NO:9 sets forth the predicted amino acid sequence of the matureprotease BspZ00056: GQTVPWGIPHVQGTAAQDAGYTGAGLKVAILDTGIDRNHEDLFANVKGGHSVFTDSANSDPFYDADGHGTHVAGTVAAVDNDLGVVGVASQAELYAVKVLNNSGSGSYAGIAEGIEWSINNGMDIINMSLGGSQSSSILKQFSDLAYEEGLLVVAAAGNSGNRGGNNDTVGYPAKYDSVIAVAAVDQNNNRATFSSTGPAVEISAPGVSILSTTPGNNYAAFNGTSMASPHVAGVAAQVWQAKPELSNVELRNLLNETAVNLGGSNQFGHGLVQSLDAIQH.

Example 4 Heterologous Expression of Serine Proteases BspAP02013,BspM02866, and BspZ00056

BspAP02013, BspM02866, and BspZ00056 proteases were produced in B.subtilis using an expression cassette consisting of the B. subtilis aprEpromoter, the B. subtilis aprE signal peptide sequence, the nativeBspAP02013, BspM02866, or BspZ00056 protease pro-peptides, the matureBspAP02013, BspM02866, or BspZ00056 proteases and a BPN′ terminator.These cassettes were cloned into the pBN based replicating shuttlevector (Babe′ et al. (1998), Biotechnol. Appl. Biochem. 27: 117-124) anda suitable B. subtilis strain was transformed with the vector.

Plasmid maps of the pBN vector containing the BspAP02013gene(pBN-BspAP02013), BspM02866 (pBN-BspM02866), and BspZ00056 gene(pBN-BspZ00056) are shown in FIGS. 1-3, respectively.

To produce BspAP02013, BspM02866, and BspZ00056, suitable B. subtilistransformants containing pBN-BspAP02013, pBN-BspM02866, or pBN-BspZ00056were cultured in 15 ml Falcon tubes for 16 hours in TSB (broth) with 10ppm neomycin, and 300 μl of the pre-cultures were added to a 500 mLflask filled with 30 mL of cultivation media (described below)supplemented with 10 ppm neomycin. The flasks were incubated for 48hours at 32° C. with constant rotational mixing at 180 rpm. Cultureswere harvested by centrifugation at 14500 rpm for 20 minutes in conicaltubes. The culture supernatants were used for assays. The cultivationmedia was an enriched semi-defined media based on MOPs buffer, with ureaas major nitrogen source, glucose as the main carbon source, andsupplemented with 1% soytone for robust cell growth.

Protein Determination by Stain Free Imager Criterion

Protein was quantified by the stain-free Imager Criterion method. Themethod is based on utilizing stain-free precast PAGE gels, where theintensity of each band will depend on amount of tryptophan residuespresent in the protein of interest. The Criterion™ TGX (Tris-Glycineextended) Stain-Free™ precast gels for PAGE include unique trihalocompounds. This allows rapid fluorescent detection of proteins with theGel Doc™ EZ imaging system. The trihalo compounds react with tryptophanresidues in a UV-induced reaction to produce fluorescence, which can beeasily detected by the Gel Doc EZ imager within gels. Reagents used inthe assay: Concentrated (10×) Laemmli Sample Buffer (Kem-En-Tec,Catalogue #42556); either 18 or 26-well Criterion TGX Strain-FreePrecast gels (Bio-Rad, Catalogue #567-8124 and 567-8125, respectively);and protein markers “Precision Plus Protein Standards” (Bio-Rad,Catalogue #161-0363). The assay was carried out as follows: 25 μlprotein sample and 25 μl 0.5M HCL was added to a 96 well-PCR plate onice for 10 min to inactivate the protease and prevent self-hydrolysis.50 μl of the acid protein mix was added to 504, sample buffer containing0.385 mg DTT in the 96 well-PCR plate. After that, the chamber wasfilled by running buffer, and the gel cassette was set. 10 μL of eachsample together with markers was load into each pocket and theelectrophoresis was started at 200 V for 35 min. Followingelectrophoresis, the gel was transferred to Imager. Image Lab softwarewas used for calculation of intensity of each band. By knowing theprotein amount and the tryptophan content of the standard sample, thecalibration curve can be made. The amount of experimental sample can bedetermined by extrapolation of the band intensity and tryptophan numbersto protein concentration. The protein quantification method was employedto prepare samples of the BspAP02013, BspM02866, and BspZ00056 proteasesused for the assays set forth in Examples 5-8.

N and C-Terminal Amino Acid Determination

In preparation for sequence confirmation, a sample of isolated BspM2866protein was subjected to a series of chemical treatments in a 10 kDaspinfilter. The sample was denatured and reduced by urea and DTTtreatment. A guanidination step was performed to convert lysines tohomoarginines to protect lysine side chains from acetylation.Acetylation reaction using iodoacetamide then modifies only theproteins' N-terminal residue. The sample was then mixed with a buffercontaining ¹⁸O water and the enzymes trypsin and chymotrypsin added fordigestion. The resulting peptides contained mixtures of ¹⁸O and ¹⁶O,except for the Carboxyl terminus which retains the native ¹⁶O. Thedigestion products were separated and analyzed using a Proxeon nano-LCsystem followed by LTQ Orbitrap (Thermo Fisher) high resolution massspectrometer. The amino acid sequence was deduced from the MS/MSfragment spectrum of the peptides and the isotopic pattern of thepeptides. A sample of BspM2866 protein expressed from plasmidpBN-BspAP02013 was analyzed as described above. The sequence of themature protein was determined to correspond to SEQ ID NO:6, 281 aminoacids, set forth above.

Example 5 Protease Activity of BspAP02013, BspM02866, and BspZ00056

The protease activity of BspAP02013(SEQ ID NO:3), BspM02866(SEQ IDNO:6), and BspZ00056 (SEQ ID NO:9) were tested by measuring thehydrolysis of dimethyl casein (DMC) substrate. The reagent solutionsused for the DMC assay were: 2.5% DMC (Sigma) in 100 mM Sodium CarbonatepH 9.5, 0.075% TNBSA (2,4,6-trinitrobenzene sulfonic acid, ThermoScientific) in Reagent A. Reagent A: 45.4 g Na₂B₄O₇.10H₂O (Merck) in 15mL 4N NaOH to reach a final volume of 1000 mL in MQ water, DilutionSolution: 10 mM NaCl, 0.1 mM CaCl₂), and 0.005% Tween-80. Proteasesupernatants were diluted in Dilution Solution to appropriateconcentration for the assay. A 96-well microtiter plate (MTP) was filledwith 95 μl DMC substrate followed by the addition of 5 μl dilutedprotease supernatant. 1004, of TNBSA in Reagent A was then added withslow mixing. Activity was measured at 405 nm over 5 min using aSpectraMax plate reader in kinetic mode at RT. The absorbance of a blankcontaining no protease was subtracted from values. The activity wasexpressed as mOD/min. The protease activity curves for BspAP02013,BspM02866, and BspZ00056 proteases are shown in FIGS. 4-6, respectively.Using the DMC assay, the specific activity of BspAP02013 was found to be60 mOD/min/ppm, of BspM02866 was found to be 39 mOD/min/ppm, and ofBspZ00056 was found to be 18 mOD/min/ppm. The specific activities ofGG36 and BPN′ proteases were found to be 54 and 23 mOD/min/ppm,respectively under the same assay conditions.

Example 6 pH Profile of BspAP02013

The pH dependence of proteolytic activity of BspAP02013 (SEQ ID NO:3)was studied using azo-casein as substrate in a 50 mMAcetate/Bis-Tris/HEPES/CHES buffer including 50 mM CaCl₂). The activitywas measured at pH between 4 to 12 with 1 pH unit increments. OneProtaxyme AK tablet (Megazyme, Ireland) was added to a glass test tubetogether with 1.9 mL of appropriate buffer and a magnet, followed bygentle hydration at 40° C. for 5 min in a temperature controlled waterbath fitted with magnetic stirrer. A 100 μL sample of freshly preparedprotease (diluted in deionised water to appropriate concentration forthe assay) was added to the prehydrated substrate and reaction wascarried out at 40° C. for 10 min. To terminate the reaction, 10 mL of a2% w/v Tris buffer, pH 12 was added, solution was mixed, and the samplewas immediately filtered through a Whatman No. 1 filter. The supernatantwas collected and the absorbance at 590 nm of the supernatant wasmeasured to quantify the product of the reaction. The absorbance from abuffer-only control was subtracted, and the resulting values wereconverted to percentages of relative activity, by defining the activityat the optimal pH as 100%. BspAP02013 was determined to maintain ≥50% ofactivity over the pH range of 8-12 under the conditions of this assay.

Example 7 Temperature Profile of BspAP02013

The temperature dependence of proteolytic activity of BspAP02013 (SEQ IDNO:3) was studied using azo-casein as substrate in a 50 mMAcetate/Bis-Tris/HEPES/CHES buffer including 50 mM CaCl₂) at pH 10. Theactivity was measured at temperatures between 30° C. and 80° C. with 10°C. increments. One Protaxyme AK tablet (Megazyme, Ireland) was added toa glass test tube together with 1.9 mL of appropriate buffer and amagnet stirrer, followed by gentle hydration at set temperatures for 5min in a temperature controlled water bath fitted with magnetic stirrer.A 100 μl sample of freshly prepared protease (diluted in deionised waterto appropriate concentration for the assay) was added to the prehydratedsubstrate and reaction was carried out at temperatures between 30° C.and 80° C. for 10 min. To terminate the reaction, 10 mL of a 2% w/v Trisbuffer pH 12 was added and solution was mixed and filtered immediatelythrough a Whatman No. 1 filter. The supernatant was collected and theabsorbance at 590 nm of the supernatant was measured to quantify theproduct of the reaction. The absorbance from a buffer-only control wassubtracted from each sample reading, and the resulting values wereconverted to percentages of relative activity, by defining the activityat the optimal temperature at 100%. BspAP02013 was determined to retain≥50% activity over a range of 55° C.−75° C., under the conditions ofthis assay.

Example 8 Stability Evaluation of Proteases

The stability of BspAP02013 (SEQ ID NO:3) and FNA (SEQ ID NO:10)proteases was determined under various conditions.

SEQ ID NO:10 sets forth the sequence of FNA protease: AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGALNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ.

Stability was tested under two stress conditions shown below bymeasuring the residual proteolytic activity following incubation at settemperatures.

1. LAS/EDTA:

0.02% LAS, 2.1 mM EDTA in 50 mM HEPES pH8, 0.005% Tween 80

2. OMO HDL:

10% OMO Klein & Krachtig liquid detergent (heat inactivated prior touse).

For stressed conditions, diluted enzyme sample was mixed in stressbuffers/detergent in a 96-well PCR plate and incubated at 30° C., 40°C., 50° C., 60° C. and 75° C. for 20 min using a Tetrad2 Thermocycler.For the unstressed condition, enzyme was assayed immediately aftermixing with stress media to establish a baseline (initial activity).Protease activity under stressed and unstressed conditions was measuredby either the hydrolysis of AAPF-pNA or DMC substrate assays describedpreviously. Percent residual activities were calculated by taking aratio of the stressed to unstressed activity at each temperature andmultiplying it by 100. The percent remaining activity for each proteaseis shown on Tables 1 and 2 for each condition run at the varioustemperatures.

TABLE 1 Stability of proteases in LAS/EDTA Incubation temperature EnzymeUnstressed 30° C. 40° C. 50° C. 60° C. 75° C. BspAP02013 100 >90 69 3330 31 FNA 100 >90 84 7 4 1

TABLE 2 Stability of proteases in 10% OMO HDL Incubation temperatureEnzyme Unstressed 30° C. 40° C. 50° C. 60° C. 75° C. BspAP02013100 >90 >90 89 11 1 FNA 100 >90 >90 71  1 1

Example 9 Comparison of BspAP02013, BspM02866, and BspZ00056 Protease toRelated Molecules Identification of Homologous Proteases

Related proteins were identified by a BLAST search (Altschul et al.,Nucleic Acids Res, 25:3389-402, 1997) using the mature protein aminoacid sequences for BspAP02013 (SEQ ID NO: 3), BspM02866 (SEQ ID NO:6),and BspZ00056 (SEQ ID NO:9) against the NCBI non-redundant proteindatabase and a subset are shown on Tables 4A, 5A, and 6A, respectively.A similar search was run against the Genome Quest Patent database withsearch parameters set to default values using the mature protein aminoacid sequences for BspAP02013 (SEQ ID NO:3), BspM02866 (SEQ ID NO:6),and BspZ00056 (SEQ ID NO:9) as the query sequence, and a subset areshown in Tables 4B, 5B, and 6B, respectively. Percent identity (PID) forboth search sets is defined as the number of identical residues dividedby the number of aligned residues in the pairwise alignment. Valuelabeled “Sequence length” on tables corresponds to the length (in aminoacids) for the proteins referenced with the listed Accession numbers,while “Aligned length” refers to sequence used for alignment and PIDcalculation.

TABLE 4A List of sequences with percent identity to BspAP02013 proteinidentified from the NCBI non-redundant protein database SequenceAlignment Accession # PID Organism Length Length WP_047973355 96Bacillus sp. LL01 377 281 YP_003427361.1/ 90 Bacillus pseudofirmus OF4379 281 WP_012957833 ERN55058/ 90 Bacillus marmarensis DSM 21297 291 281WP_022626565.1 WP_026690432 85 Bacillus aurantiacus 377 280 BAD02409 75Bacillus sp. KSM-LD1 404 279 WP_035661169 73 Bacillus akibai JCM 9157378 281 WP_027963976 71 Halalkalibacillus halophilus 372 279WP_027965007 71 Halalkalibacillus halophilus 377 280 WP_026475840 70Alkaliphilus transvaalensis 382 279 WP_017185212.1 65 Alkalibacillushaloalkalphilus 374 278 ADC49870 58 B pseudofirmus 374 280 BAD21128 58Bacillus sp KSM-LD1 SB 377 280 AAC43580 58 Bacillus sp. SprC 378 280CAJ70731 57 Bacillus licheniformis 379 280 BAD11988 57 Bacillus sp.KSM-LD1 SA 376 280 YP_003972439 57 B atrophaeus 382 280 WP_006636716 56B sonorensis 378 280 AAC43581 56 Bacillus sp SprD 379 280 BAN09118 56 Bsubtilis 381 280 CAA24990 56 Bacillus amyloliquefaciens 376 280 AAA2221255 B alcalophilus 380 275 P29600 55 Bacillus lentus 269 275 BAD63300 55B clausii 380 275 WP_010333625 55 B mojavensis 381 275 WP_010329279 55 Bvallismortis 381 280 BAA06157 54 Bacillus sp. Sendai 382 274 ADN04910 54B circulans 275 273 AFP23380.1 54 B lehensis 276 273 WP_007497196 54 Bstratosphericus 383 273 ADK11996 54 B pumilus 383 273 AGC81872.1 54 Bmethylotrophicus 382 280 CAA74536 53 B subtilis str168 381 280 ABY2585653 G stearothermophilus 382 280

TABLE 4B List of sequences with percent identity to BspAP02013 proteinidentified from the Genome Quest database Sequence Alignment Patent ID #PID Organism Length Length JP2003325186-0001 74.6 Bacillus sp. 404 279JP2004313043-0024 59.6 Bacillus sp. KSM-9865 379 280 U.S. Pat. No.5,275,945-0002 59.3 Bacillus sp. 377 280 JP2012524542-0052 59.1Alkaliphilus transvaalensis 274 279 JP2008022828-0032 58.9 Bacillus sp.352 280 JP2004154003-0007 58.6 Bacillus sp. KSM-LD1 276 280 U.S. Pat.No. 5,677,163-0001 58.2 Bacillus sp. 275 280 JP2013500714-0412 57.9Bacillus licheniformis 274 280 JP2013500714-0403 57.9 Bacillusmojavensis 274 280 WO2013159032-0004 57.9 Bacillus licheniformis 274 280WO2012139964-0002 57.9 Bacillus sp. 274 280 U.S. Pat. No. 8,110,391-000957.9 Bacillus licheniformis 274 280 CN101215534 57.5 B. licheniformis;YP1A, CCTCC NO: M207021 379 280 W002077289-0009 58.6 Bacillus sp. 372273 JP2013500005-0025 57.1 Bacillus licheniformis 274 280US20050113273-0016 57.1 Bacillus licheniformis 274 280 U.S. Pat. No.8,076,107-0001 57.1 Bacillus licheniformis 379 280 KR20080017039-000557.1 Bacillus licheniformis 379 280

TABLE 5A List of sequences with percent identity to BspM02866 proteinidentified from the NCBI non-redundant protein database SequenceAlignment Accession # PID Organism Length Length WP_026690432 86Bacillus aurantiacus 377 281 YP_003427361.1/ 81 Bacillus pseudofirmusOF4 379 280 WP_012957833 WP_047973355 81 Bacillus sp. LL01 377 281ERN55058/ 80 Bacillus marmarensis DSM 21297 291 280 WP_022626565.1WP_035661169 75 Bacillus akibai JCM 9157 378 280 BAD02409 75 Bacillussp. KSM-LD1 404 279 WP_026475840 71 Alkaliphilus transvaalensis 382 279WP_027965007 71 Halalkalibacillus halophilus 377 280 WP_027963976 70Halalkalibacillus halophilus 372 278 WP_017185212.1 66 Alkalibacillushaloalkaliphilus 374 278 AAC43580 61 Bacillus sp. SprC 378 278 AAC4358160 Bacillus sp SprD 379 272 BAD21128 60 Bacillus sp KSM-LD1 SB 377 272BAD11988 59 Bacillus sp. KSM-LD1 SA 376 272 CAJ70731 58 Bacilluslicheniformis 379 272 WP_006636716 56 B sonorensis 378 272 ADC49870 56 Bpseudofirmus 374 274 BAD63300 55 B clausii 380 274 YP_003972439 55 Batrophaeus 383 272 AAA22212 54 B alcalophilus 380 274 P29600 54 Bacilluslentus 269 274 BAA06157 53 Bacillus sp. Sendai 382 274 ADN04910 53 Bcirculans 275 279 AFP23380.1 53 B lehensis 276 279 WP_007497196 53 Bstratosphericus 383 279 ADK11996 53 B pumilus 383 279 AGC81872.1 53 Bmethylotrophicus 382 272 BAN09118 52 B subtilis 381 272 CAA24990 52Bacillus amyloliquefaciens 376 272 ABY25856 52 G stearothermophilus 383272 WP_010333625 51 B mojavensis 381 272 WP_010329279 51 B vallismortis381 272 CAA74536 50 B subtilis str168 381 272

TABLE 5B List of sequences with percent identity to BspM02866 proteinidentified from the Genome Quest database Sequence Alignment Patent ID #PID Organism Length Length JP2003325186-0001 74.9 Bacillus sp. 404 279U.S. Pat. No. 5,677,163-0001 61.2 Bacillus sp 275 278 JP2004313043-002260.7 Bacillus sp. KSM-9865 275 272 U.S. Pat. No. 5,677,163-0007 60.3Bacillus sp. 276 272 JP2004154003-0007 60.3 Bacillus sp. KSM-LD1 276 272JP2004313043-0021 59.9 Bacillus sp. KSM-KP43 275 272 JP2013500714-041658.5 Bacillus licheniformis 269 272 KR20080017039-0002 58.5 Bacilluslicheniformis 379 272 U.S. Pat. No. 8,530,218-0052 56.6 Alkaliphilustransvaalensis 274 279 U.S. Pat. No. 5,371,008 58.1 Bacilluslicheniformis; (carlsbergensis) 275 272 CN101215534 58.1 B.licheniformis; YP1A, CCTCC NO: M207021 379 272 WO9739130 58.1 Bacilluslicheniformis; strain PWD-1 379 272 WO2011014278 57.7 Bacilluslicheniformis ATCC 14580 274 272 WO2011009859 57.7 Bacillusamyloliquefaciens 274 272

TABLE 6A List of sequences with percent identity to BspZ00056 proteinidentified from the NCBI non-redundant protein database SequenceAlignment Accession # PID Organism Length Length WP_026690432 86Bacillus aurantiacus 377 281 YP_003427361.1/ 81 Bacillus pseudofirmusOF4 379 279 WP_012957833 WP_047973355 81 Bacillus sp. LL01 377 280ERN55058/ 80 Bacillus marmarensis DSM 21297 291 280 WP_022626565.1BAD02409 78 Bacillus sp. KSM-LD1 404 278 WP_035661169 75 Bacillus akibaiJCM 9157 378 280 WP_026475840 71 Alkaliphilus transvaalensis 382 279WP_027965007 71 Halalkalibacillus halophilus 377 280 WP_027963976 70Halalkalibacillus halophilus 372 278 WP_017185212.1 67 Alkalibacillushaloalkaliphilus 374 280 WP_010192403.1 61 Bacillus sp. m3-13 381 279AAC43580 60 Bacillus sp. SprC 378 279 BAD21128 58 Bacillus sp KSM-LD1 SB377 279 BAD11988 58 Bacillus sp. KSM-LD1 SA 376 279 AAC43581 57 Bacillussp SprD 379 279 CAJ70731 57 Bacillus licheniformis 379 279 WP_00663671657 B sonorensis 378 280 ADC49870 56 B pseudofirmus 374 279 YP_00397243956 B atrophaeus 382 279 BAD63300 54 B clausii 380 274 ADN04910 54 Bcirculans 275 272 AFP23380.1 54 B lehensis 276 272 WP_007497196 54 Bstratosphericus 383 272 ADK11996 54 B pumilus 383 272 AAA22212 53 Balcalophilus 380 274 P29600 53 Bacillus lentus 269 274 BAN09118 53 Bsubtilis 381 279 CAA24990 53 Bacillus amyloliquefaciens 376 279WP_010333625 53 B mojavensis 381 279 BAA06157 52 Bacillus sp. Sendai 382274 AGC81872.1 52 B methylotrophicus 382 279 ABY25856 51 Gstearothermophilus 382 279 WP_010329279 51 B vallismortis 381 279CAA74536 51 B subtilis str168 381 279

TABLE 6B List of sequences with percent identity to BspZ00056 proteinidentified from the Genome Quest database Sequence Alignment Patent ID #PID Organism Length Length JP2003325186-0001 78.1 Bacillus sp. 404 278WO9406915-0001 60.2 Bacillus sp 275 279 JP2004313043-0022 58.8 Bacillussp. KSM-9865 275 279 JP2004065171-0007 58.4 Bacillus sp. KSM-LD1 376 279WO9855634 58.1 Bacillus licheniformis 274 279 JP2004313043 58.1 Bacillussp. KSM-KP43 379 279 JP2013500714-0403 57.7 Bacillus mojavensis 274 279U.S. Pat. No. 7,087,415-0014 57.7 Bacillus licheniformis 379 279CN101215534 57.4 B. licheniformis; YP1A, CCTCC NO: M207021 379 279WO9739130 57.4 Bacillus licheniformis; strain PWD-1 379 279 WO201100985957.0 Bacillus amyloliquefaciens 274 279 U.S. Pat, No. 8,530,218-005256.8 Alkaliphilus transvaalensis 274 278

Alignment of Homologous Sequences

An alignment of the mature protein amino acid sequences for BspAP02013(SEQ ID NO:3), BspM02866 (SEQ ID NO:6), and BspZ00056 (SEQ ID NO:9) withthe sequences of the mature forms of various subtilisins from Tables 4A,5A, and 6A is shown in FIG. 7. The sequences were aligned using CLUSTALWsoftware (Thompson et al., Nucleic Acids Research, 22:4673-4680, 1994)with the default parameters. A phylogenetic tree for amino acidsequences of the mature forms of the subtilisins from FIG. 7 was builtusing the Geneious Tree builder program (Biomatters Ltd.) and is setforth in FIG. 8. The sequences were entered in the Vector NTI Advancesuite and a Guide Tree was created using the Neighbor Joining (NJ)method (Saitou and Nei, Mol Biol Evol, 4:406-425, 1987). The treeconstruction was calculated using the following parameters: Kimura'scorrection for sequence distance and ignoring positions with gaps. MEGA6was used to display the tree shown in FIG. 8.

Example 10 Unique Features of BspAP02013-Clade Subtilisins

A structure based alignment of the following proteases: BspAP02013 (SEQID NO:3), BspM02866 (SEQ ID NO:6), BspZ00056 (SEQ ID NO:9), B.pseudofirmus OF4 (NCBI Accession No. YP_003427361.1), BPN′ subtilisinfrom B. amyloliquefaciens (pdb entry 2STI), and B. lentus subtilisin(pdb entry 1JEA), was performed using the “align” option in theMolecular Operating Environment (MOE) software (Chemical ComputingGroup, Montreal, Quebec, Canada) to look for structural similarities(FIG. 9). The alignment applies conserved structural motifs as anadditional guide to conventional sequence alignment. This alignment wasperformed using standard program defaults present in the 2012.10distribution of MOE.

As shown in FIG. 9, the structural alignment of subtilisins BspAP02013,BspM02866, BspZ00056, and YP_003427361.1 (B. pseudofirmus OF4) sequencesshow a common pattern of three insertions relative to the sequences ofsubtilisins: BPN′ from B. amyloliquefaciens and subtilisin from B.lentus, for which three dimensional structures are available (pdbentries 2ST1 and 1JEA, respectively). The numbering of residues in the1JEA and 1CSE structures is with respect to subtilisin BPN′; while thenumbering of residues for BspAP02013 and all other proteases shown isthe consecutive linear sequence. The insertions follow residues L42, N56and T158 in BPN′ subtilisin.

It can be seen that the BspAP02013-clade subtilisins can becharacterized by a common motif over the sequence that begins with thecatalytic Aspartic acid (D32) of the serine protease triad and ends withthe catalytic Histidine (H68) of the catalytic triad according toBspAP02013 numbering. The motif includes two insertions each adding 2amino acid residues that can be seen in FIG. 9 (box).

These insertions are expected to occur in two loops common to theoverall tertiary fold of subtilisin enzymes. The location of theseinsertions are illustrated in FIG. 10 using the known structure ofsubtilisin BPN′ as a reference. The motif segment is highlighted inblack using the BPN′ subtilisin structure as a reference (in lightgray). The catalytic Aspartic acid and Histidine residue side chains, ofthe catalytic triad common to all serine proteinases are shown assticks. The segment that connects the catalytic Aspartic Acid (D32) andHistidine (H68) of the subtilisin catalytic triad comprises two loopsand the outermost strand of the central beta sheet. This strand includesthe GGXS portion of the BspAP02013-clade motif and is indicated by anarrow in FIG. 10. The two motif insertions are proposed to occur inloops indicated by the arrows. Insertion 1 enlarged the first loop andinsertion 2, the second loop. The loop containing Insertion 1 occurs ina loop extending from the catalytic Aspartic acid to a beta strand atthe edge of the beta sheet GGXS strand. The loop containing insertion 2follows the GGXS strand and leads into the catalytic Histidine.

The BspA02013-clade motif can be characterized by Motif 1: DTGIXXXHXDLXXXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:11), wherein X is any amino acid andcollectively can indicate regions of insertion and deletion. In theinstances shown in FIG. 9, the BspA02013-clade motif can becharacterized by Motif 2: DTGIXXXHXDLXaXXXXGGX SVFTDSXXXXXXXDXXGH (SEQID NO:12), wherein X is any amino acid and X_(a) is T, S or F, wherein Xand X_(a) can collectively indicate regions of insertion and deletion.In the instances shown in FIG. 9, the BspA02013-clade motif can becharacterized by Motif 3: DTGIXXXHX DLXXXXXGGXSVFTDSXXX_(b)XXXXDXXGH(SEQ ID NO:13), wherein X is any amino acid and X_(b) is S or R. In theinstances shown in FIG. 9, the BspA02013-clade motif can becharacterized by Motif 4: DTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXX_(b)XXXXDXXGH(SEQ ID NO:14), wherein X is any amino acid, X_(a) is T, S or F, andX_(b) is S or R. The BspAP02013, BspM02866, BspZ00056, YP_003427361(B.pseudofirmus OF4) and ERN55058/WP_022626565 (B. marmarensis) subtilisinswhich have been identified as BspAP02013-clade subtilisins based onshared sequence motifs outlined in FIG. 9, also cluster together in thephylogenetic tree built using various bacterial subtilisins shown onFIG. 8, with a distance of 0.08.

The amino acid identity across the mature forms of various subtilisinsfrom Tables 4A, 5A, and 6A is shown on Table 7 below, wherein thepercent amino acid identity is calculated over the 281 residues of theBspAP02013 mature sequence.

TABLE 7 Percent amino acid sequence identity Mature enzyme 1 2 3 4 5 6 78 9 10 11 12  1. BspAP02013 80.8 85.4 96.1 90.4 89.7 69 84.7 70.8 73 7470.1  2. BspM02866 80.8 86.8 80.8 80.4 79.7 70.5 86.1 70.8 74.7 74.468.3  3. BspZ00056 85.4 86.8 85.8 86.8 86.1 71.2 98.2 71.9 74.7 77.271.9  4. B-spLL01_WP_047973355 96.1 80.8 85.8 89.3 88.6 68.7 85.1 69.874 75.1 69.4  5. B_pseudofirmus_YP_003427361 90.4 80.4 86.8 89.3 99.369.8 86.5 70.8 74 75.4 70.8  6. B_marmarensis_WP_022626565_ 89.7 79.786.1 88.6 99.3 69 85.8 70.8 73.7 75.1 70.5 ERN55058  7.A_transvaalensis_WP_026475840 69 70.5 71.2 68.7 69.8 69 70.5 63.3 69 7365.1  8. B_aurantiacus_WP_026690432 84.7 86.1 98.2 85.1 86.5 85.8 70.571.9 75.4 77.2 71.2  9. H_halophilus_WP_027965007 70.8 70.8 71.9 69.870.8 70.8 63.3 71.9 68.3 68.7 68.7 10. B_akibai_WP_035661169 73 74.774.7 74 74 73.7 69 75.4 68.3 76.2 66.5 11. B_sp_BAD02409 74 74.4 77.275.1 75.4 75.1 73 77.2 68.7 76.2 68.6 12. H_halophilus_WP_027963976 70.168.3 71.9 69.4 70.8 70.5 65.1 71.2 68.7 66.5 68.6

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein can be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-23. (canceled)
 24. A method of cleaning, comprising contacting asurface or an item in need of cleaning with a composition comprising asurfactant and a polypeptide or active fragment thereof of theBspAP02013-clade; and optionally further comprising the step of rinsingsaid surface or item after contacting said surface or item with saidcomposition.
 25. The method of claim 24, wherein said item is dishwareor fabric. 26-36. (canceled)
 37. The method of claim 24, wherein thepolypeptide or active fragment thereof comprises one or more motifselected from: (i) DTGIXXXHXDLXXXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:11)motif, wherein the initial D is the active site Aspartic acid, theterminal H is the active site Histidine, and X is any amino acid; (ii)DTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXXXXXXDXXGH (SEQ ID NO:12) motif, whereinthe initial D is the active site Aspartic acid, the terminal H is theactive site Histidine, X is any amino acid, and X_(a) is T, S or F orX_(a) is S or F; (iii) DTGIXXXHXDLXXXXXGGXSVFTDSXXX_(b)XXXXDXXGH (SEQ IDNO:13) motif, wherein the initial D is the active site Aspartic acid,the terminal H is the active site Histidine, X is any amino acid, andX_(b) is S or R or X_(b) is S; (iv)DTGIXXXHXDLX_(a)XXXXGGXSVFTDSXXX_(b)XXXXDXXGH (SEQ ID NO:14) motif,wherein the initial D is the active site Aspartic acid; the terminal His the active site Histidine; X is any amino acid; X_(a) is T, S or F orX_(a) is S or F; and X_(b) is S or R or X_(b) is S; (v)DTGIXXXHXDLXANVXGGXSVFTDSANXDPFXDXXGH (SEQ ID NO:15) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid; (vi) HXDLXANVXGGXS (SEQID NO:16) motif, wherein the initial D is the active site Aspartic acid,the terminal GGXS is in the outermost strand of the central beta sheet,and X is any amino acid; (vii) GGXSVFTDSANXDPFXD (SEQ ID NO:17) motif,wherein the initial GGXS is in the outermost strand of the central betasheet and X is any amino acid; (viii) HXDLX_(a)ANVXGGXS (SEQ ID NO:18)motif, wherein the initial D is the active site Aspartic acid; theterminal GGXS is in the outermost strand of the central beta sheet;X_(a) is T, S or F or X_(a) is S or F; and X is any amino acid; (ix)GGXSVFTDSANX_(b)DPFXD (SEQ ID NO:19) motif, wherein the initial GGXS isin the outermost strand of the central beta sheet, X is any amino acid,and X_(b) is S or R or X_(b) is 5; (x)DTGIXXXHXDLXXXXXGGXSVFXDXXXXXXXXDXXGH (SEQ ID NO:22) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid; (xi)DTGIXXXHXDLXXNVXGGXSVFXDXXNXDPXXDXXGH (SEQ ID NO:23) motif, wherein theinitial D is the active site Aspartic acid and the terminal H is theactive site Histidine, and X is any amino acid; and (x) an amino acidsequence having at least 80% amino acid sequence identity to an aminoacid sequence of SEQ ID NO:3, 6, or
 9. 38. The method of claim 37,wherein the polypeptide or active fragment thereof does not compriseWP_012957833, ERN55058, WP_022626565, or WP_026690432, or optionallyWP_047973355.
 39. The method of claim 24, wherein the surfactant isselected from the group consisting of an anionic surfactant, a cationicsurfactant, a zwitterionic surfactant, an ampholytic surfactant, asemi-polar non-ionic surfactant, and a combination thereof.
 40. Themethod of claim 24, wherein the composition is a detergent composition.41. The composition of claim 40, wherein the detergent composition isselected from the group consisting of a laundry detergent, a fabricsoftening detergent, a dishwashing detergent, and a hard-surfacecleaning detergent.
 42. The method of claim 24, wherein said compositionfurther comprises at least one calcium ion and/or zinc ion; at least onestabilizer; from about 0.001% to about 1.0 weight % of said subtilisinor recombinant polypeptide; at least one bleaching agent; at least oneadjunct ingredient; and/or one or more additional enzymes or enzymederivatives selected from the group consisting of acyl transferases,alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases,aryl esterases, beta-galactosidases, carrageenases, catalases,cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases, endo-beta-mannanases, esterases, exo-mannanases,galactanases, glucoamylases, hemicellulases, hyaluronidases,keratinases, laccases, lactases, ligninases, lipases, lipoxygenases,mannanases, oxidases, pectate lyases, pectin acetyl esterases,pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases,phospholipases, phytases, polygalacturonases, proteases, pullulanases,reductases, rhamnogalacturonases, beta-glucanases, tannases,transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases,xylosidases, metalloproteases, additional serine proteases, andcombinations thereof.
 43. The method of claim 24, wherein saidcomposition contains phosphate or is phosphate-free and/or containsborate or is borate-free.
 44. The method of claim 24, wherein saidcomposition is a granular, powder, solid, bar, liquid, tablet, gel,paste or unit dose composition.
 45. The method of claim 24, wherein saidcomposition is formulated at a pH of from about 8 to about 12.